WO2015048098A1 - Diagnostic methods for infectious disease using endogenous gene expression - Google Patents

Diagnostic methods for infectious disease using endogenous gene expression Download PDF

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WO2015048098A1
WO2015048098A1 PCT/US2014/057164 US2014057164W WO2015048098A1 WO 2015048098 A1 WO2015048098 A1 WO 2015048098A1 US 2014057164 W US2014057164 W US 2014057164W WO 2015048098 A1 WO2015048098 A1 WO 2015048098A1
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assay
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Gregory STORCH
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Washington University
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Priority to US16/797,733 priority patent/US11286529B2/en

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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Virus and bacteria interact with different pattern recognition receptors in circulating blood leukocytes, triggering specific host transcriptional programs mediating immune response.
  • Unique transcriptional signatures can be defined that discriminate viral from bacterial causes of certain infectious disorders.
  • Viral infections can be a cause of Fever Without an Apparent Source (FWS) in young children.
  • Host transcriptional profiles can be useful if a pathogen is not detected or if the pathogenic role of a detected agent is in question .
  • Circulating blood leukocytes can react, to pathogens by recognizing pathogen-specific molecular patterns through pattern recognition receptors leading to up or down-regulation of the expression of host genes associated with immune functions (Takeuchi, O., et al. 2010, Cell 140: 805-820 and Thompson, M. ., et l. 201 1 , Viruses 3: 920-940).
  • Host Host
  • transcriptional programs can be.- differentially activated with different pathogens (Pasehos, K., et al. 2010, Trends Microbiol J 8(10):439- 47).
  • PCT/US2007/075713 (WO 2008/024642 A2) of Banchereau, J.F., ei al., describes specific gene expression biomarkers for the differential diagnosis of >$ aureus, E. cali f and influenza infections.
  • PCT/US.2 10/046042 (WO 201 1/066008 A2) of Banchereau, J.F., ei al, describes blood transcriptional signatures of active versus laten t mycobaeterium tuberculosis infections.
  • the laboratory tests were white blood ceil count, serum C-reactive protein and the presence of >70 white blood cells in urinalysis. Their diagnostic methods. do not include an analysis of changes in host gene expression.
  • US Application number 11/268373 (US20080020379 l) of Agan, B. ., et al provides a specific set of host gene expression markers, for identification of pathogenic infections with 47 genes as the minimal number of genes to classify febrile versus non-febrile patients, Analysis of host transcriptional profiles has been applied to the diagnosis of inflammatory and hematological diseases. For example in Aliantaz, et al, 2007, J, Exp. Med. 204: 2131 -2144; Aaroe, I, et al 2010, Bre t Cancer Res !2:R7; Al.kadeh, A.A., et l 2000, Nature 403: 5(53-51 1 ; and ChaussabeL D., ei al. 201. 1 , "Blood Transcriptional
  • the present inventor has developed a prognostic model for diagnosis of a pathogen- associated or pathogen-based disease in a subject.
  • the expression levels of endogenous prognostic genes can. provide clinically beneficial assays as well as biological insights.
  • testing expression le vels of endogenous prognostic genes can be supplemented with assays to determine presence, absence, and/or quantity of a pathogen.
  • the present inventor discloses methods of di agnosis of a pathogen-associated disease in a subject Additionally disclosed are methods of distinguishing a viral -caused infection from a bacterial-caused infection or a combination thereof. Additionall disclosed arc methods of diagnosis of a viral pathogen-associated disease and methods of diagnosis of Fever Without an Apparent Source (FWS),
  • FWS Apparent Source
  • the present teachings include: host transcriptional profiles that can distinguish symptomatic from asymptomatic viral infection; virus-specific transcriptional profiles for DNA and RNA viruses that cause systemic infection; and viral and bacterial -specific transeripiional profiles that can distinguish between infections caused by different pathogens-
  • diagnostic methods of the present teachings include detecting transcriptional changes in multiple endogenous genes in multiple pathways in febrile children who are infected with D A viruses. RNA viruses, or bacteria.
  • Host transcriptional analysis can be a clinically relevant testing method, and additionally, can supplement pathogen-based nucleic acid amplification assays. Host transcriptional analysis can provide results associated with etiology. In some embodiments, host transcriptional analysis can provide results associated with etiology even when no pathogens are confirmed from the direct detection testing for microbial pathogens.
  • host blood transcriptional signatures associated with instance of infectious etiology can be defined in a subject with FWS, such as a young child with FWS, In some embodiments, host blood transcriptional signatures can be of increased accuracy and predictive value than white blood ceil count-based criteria alone in
  • host blood transcriptional signatures can be a supplement to tests that detect the presence of a possible pathogen but do not address its pathogenic role in the subject.
  • some genes of the Interfero Signaling Pathway can be uniquely activated in a febrile child with a viral infection
  • some genes of the Integrin Signaling Pathway can be uniquely activated in a child with a bacterial infection.
  • host transcriptional profiles can be used to classify a febrile child with a viral or a bacterial infection with more accurac than a blood white blood cell (WBC) count.
  • WBC blood white blood cell
  • the present teachings include methods of diagnosis of a pathogen-based disease in a subject, and methods of determining etiology of a pathogen- based disease in a subject, in various configurations, these methods comprise: a) providing at least one biological sample from a human subject; b) determining presence, absence and/or quantity of a bacterial pathogen, a viral pathogen, or a combination thereof, by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DMA detection test, a pathogen RNA detection test, or a combination thereof; and c) determining in the at least one sample, expression levels of at least one endogenous gene in which aberrant expression levels are associated with infection with a pathogen, by a microarray hybridization assay, an NA-seq assay, a polymerase chain reaction assay, a LAMP assay, a Hgase chain reaction assay, a Southern blot assay, a Northern blot as
  • the at least one endogenous gene can consist of one gene,. In some configurations, the at least one endogenous gene can consist of two genes. In some configurations, the at least one endogenou gene can consist of three genes, in some configurations, the at least one endogenous gene can consist of four genes, in some configurations, the at least one endogenous gene can consist of five genes. In some configurations, the at least one endogenous gene can consist of six genes, in some configurations, the at least one endogenous gene can consist of seven genes, in some configurations, the at. least one endogenous gene can consist of eight genes. In some configurations, the. at least one endogenous gene can consist of nine genes. In some configurations, the at.
  • the at least one endogenous gene can consist of ten genes. In some configurations, the at least one endogenous gene can consist, of eleven genes. In some configurations, the at least, one endogenous gene can consist of twelve genes. In some configurations, the at least one endogenous gene can consist of thirtee genes, i some configurations, the at least one endogenous gene can consist of fourteen genes, in some configurations, the at. least, one endogenous gene can consist of fifteen genes. In some configurations, the at least, one endogenous gene can. consist of sixteen genes. In some configurations, the at least one endogenous gene can consist of seventeen genes, in some configurations, the at least one endogenous gene can consist of eighteen genes.
  • the at least one endogenous gene can consist of nineteen genes. In some configurations, the at least one endogenous gene can. consist of twen t genes, in some configurations, the at least one endogenou gene can consist of 21. genes. In some configurations, the at least one endogenous gene can consist of 22 genes. In some configurations, the at least one endogenous gene can consist of 23 genes. In some configurations, the at least one endogenous gene can consist of 24 genes. In some configurations, the at. least, one endogenou gene can consist of 25 genes. In some configurations, the at least one endogenous gene can consist of 26 genes. In some configurations, the at least one endogenous gene can consist of 27 genes.
  • the at least one endogenous gene can consist of 28 genes. In some configurations, the at least one endogenous gene can consist of 29 genes. I some configurations, the at least one endogenous gene can consist of 30 genes. In some configurations, the at least one endogenous gene can consist of 31 genes. In some configurations, the at least one endogenous gene can consist of 32 genes, hi some configurations, the at least one endogenous gene can consist of 33 genes.
  • the disease can be Fever Without an Apparent Source.
  • the pathogen can be a virus.
  • the virus can be selected from the group consisting of an adenovirus, an enterovirus, a human herpesvirus 6 (HBV-6) and a rhiiiovims.
  • the pathogen can be a bacterium.
  • the bacterium can be selected from the group consisting of an Escherichia coil, a Staphylococcus aureus a Streptococcus
  • the at least one endogenous gene can be selected from the group consisting of IFI27, ISO 15, OTOF, 1FIT3, ITGAM and 1TGAX.
  • the at; least one gene can comprise, consist essentially of, or consist of OTOF and JTGAX.
  • the at least one gene can comprise, consist essentially of, or consist of IFI27 and ITGAM.
  • the at least one endogenous gene can be selected from the group consisting of IFI27, ISO 15, OTOF, 1FIT3, ITGAM and 1TGAX.
  • the at; least one gene can comprise, consist essentially of, or consist of OTOF and JTGAX.
  • the at least one gene can comprise, consist essentially of, or consist of IFI27 and ITGAM.
  • the at least one endogenous gene can be selected from the group consisting of IFI27, ISO 15, OTOF, 1FIT3, ITGAM and 1TGAX.
  • the at; least one gene can comprise, consist essentially of, or consist of OTOF and JT
  • determining expression levels ca comprise, consist essentially of, or consist of a real time polymerase chain reaction assay, a reverse transcriptase polymerase chain reaction assay, or a combination thereof.
  • the determining expression levels can comprise, consist essentially of, or consist of an oligonucleotide array assay, a probe hybridization assay, a gene expression array assay, a cDNA mieroarray hybridization assay or a
  • the microarray hybridization assay can comprise, consist essentiall of, or consist of from .1 to 47,300 probes, or about 47,300 probes, In some configuration , the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 5700 probes, or about 5700 probes. In some
  • the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 50 probes, or about 50 probes. In some con figurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 10 probes, or about 10 probes, in some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of between 18 and 33 probes, or about 33 probes, in some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from I to 260 probes, or about 260 probes.
  • the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 1321 probes, or about 1321 probes, in some configurations, the .microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 18 probes, or about I S probes. In some configurations, the microarray hybridization assay can comprise, consist essentially of or consist of from 1. to 22 probes, or about 22 probes.
  • the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 33 probes, or about 33 probes, in some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 260 viral response-specific probes or about 260 viral response-specific probes and from 1 to 1.321 bacterial response-specific probes or about I 321 bacteria! response-specific probes, in some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from to 3467 HHV-6 response- specific probes or about 3467 HHV-6 response-specific probes.
  • the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 464.HHV-6 response-specific probes or about 464 HHV-6 response-specific probes. In some configurations, the microarray hybridization assay can. comprise, consist essentially of, or consist of from 1 to 798 HHV-6 response-specific probes or about 798 HHV-6 response- specific probes. In some configurations, the microarray hybridization assay can comprise, consist essentiall of, or consist of from 1 to 5604 adenovirus response-specific probes or about 5604 adenovirus response-specific probes.
  • the microarray h bridization assay can comprise, consist essentially of, or consist of from I to 2078 adenovirus response- specific probes or about 2078 adenovirus response-specific probes.
  • the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 847 adenovirus response-specific probes or about 847 adenovirus response-specific probes, in some configurations, the microarray hybridization assay can comprise, consist essential ly of, or consist of from 1 to 4184 enterovirus response-specific probes or about 4184 enterovirus response-specific probes.
  • the microarray hybridization assay can comprise, consist essentially of, or consist of from 1.
  • the microarray hybridization assay ca comprise, consist essentially of, or consist of from 1 to 678 enterovirus response-specific probes or about 678 enterovirus response-specific probes. In some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1. to 1.234 bacterial response-specific probes or about 1234 bacterial response-specific probes. In some configurations, the microarray hybridization, assay can comprise, consist essentially of or consist of from I to 1939 bacterial response-specific probes or about. 1 39 bacterial response-specific probes.
  • the at least one biological sample can be selected from the group consisting of peripheral blood mononuclear cells, a nasopharyngeal sample, a urine sample, a blood sample, a lumbar puncture sample, bodil fluid, a biopsy sample, a tissue sample and a combination thereof.
  • the at least one biological sample can comprise, consist essentially of, or consist of a peripheral blood sample.
  • the at least one biological sample can comprise, consist essentially of, or consist of peripheral blood mononuclear cells, in some configurations, the at least one endogenous -gene can be selected from the group consisting of Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses Pathway genes, TREMI Signaling Pathway genes, Toll-like Receptor Signaling Pathway genes, Natural Killer Cell Signaling Pathway genes, Interferon Signaling Pathway genes, Activation, of Interferon Regulatory Factors by Cytosolic Pattern Recognition Receptors Pathway genes, Integra Signaling Pathway genes and a combination thereof, in.
  • Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses Pathway genes TREMI Signaling Pathway genes, Toll-like Receptor Signaling Pathway genes, Natural Killer Cell Signaling Pathway genes, Interferon Signaling Pathway genes, Activation, of Interferon Regulatory Factors by Cytosolic Pattern Recognition Receptors Pathway genes, Integra Signal
  • the at least one endogenous gene can comprise, consist essentially of, or consist of an interferon Signaling Pathway gene. In some configurations, the at least one endogenous gene can comprise, consist essentially of, or consist of an tegrin Signaling Pathway geae.
  • the human subject can be a human child, such as, without limitation, a human subject between 0 to 36 months of age. In some configurations, the human subject can be a human child, such as, without limitation, human subject between 2 to 36 months of age.
  • the present teachings include methods of distinguishing a viral-caused infection from a bacterial-caused infection or a combination thereof in a subjec in various configurations, these methods can comprise: a) obtaining at least one biological sample from a human subject; b) determining presence, absence and/or quantity of a viral pathogen by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DNA detection test, a pathogen RNA detection test or a combination thereof; c) determining presence, absence and/or quantity of a bacterial pathogen by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DNA detection test, a pathogen RNA detection test or a combination thereof; and.
  • the at least one gene associated with, a bacterial infection can be selected from the group consisting of FYN, CD247, EITPR3, CD3, ZAP70, PiXGl , PRKCFi, LCK, i T, PRKCQ ⁇ ⁇ , RHOU, GNA13, PPPIR12A, RHOTl, FCER IG, LYN, RALB, GNAQ, MARC S, TGM2,
  • the at least one gene associated with a bacterial infection can be selected from the group consisting of RHOU
  • FCERI G FCERI G, LYN, RALB, GNAQ, ARCKS, TGM2, ARHGEFI ⁇ , ⁇ 2A, EP300, MYL9, CREB5, FCGR2 .
  • GNGl 0, GNGl 1 , CTQB, NOD2, TLR2, TLRI , RNASEL, C5 .RL TLR4, MYD88, PIK3CB, C3AR1 , TLR6, CASPl , TLR5, NLRC4, TLR8, ILI B, PPPIRl , ZYX, MYL 12A, VASP, ITGA2B, ITGB5, VCL, ITGB3, MYLK, ASAP I, STGAM, HOAX, FCGR3B, SIGLEC9, FCERIG, XAK2, CASP5 and a combination thereof, wherein the at least one gene has an aberrant level of expression of at least a 1.5 fold increase compared to a control level in some configurations, the at least one gene associated with
  • the at least one gene has an aberrant level of expression of at least a 1.5 fold decrease compared to a. control level, m some configurations, the at least one gene associated with a bacterial infection, can be an Annexin A3 gene.
  • the patient can be treated by routine methods such as administratio of an antibiotic, which can be any antibiotic known to skilled artisans.
  • the patient can be treated by routine methods such as administration of an anti-viral drug, which can. be any anti-viral drag known to skilled artisans,
  • the present teachings include methods of diagnosing or determining etiology of Fever Without an Apparent Source in a subject I» various configurations, these methods can comprise: a) providing at least one biological sample from a human subject; b) determining, presence, absence and/or quantity of a bacterial pathogen, viral pathogen, or a combination thereof, by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DNA detection test, a pathogen RNA detection test, o.r a combination thereof; c) determinin in the sample, expression levels of at least one endogenous gene associaied with aberrant expression levels resulting from inlection with the pathogen, by a microarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay
  • the patient can be treated, by routine methods such as administration of an antibiotic.
  • the patient can be treated by routine methods such as administration of an anti-viral drug, in various configurations, the sample ca be a. peripheral blood sample, a nasopharyngeal sample, a urine sample, a blood sample, a lumbar puncture sample, a bodily fluid, a biopsy sample, a tissue sample or a combination thereof
  • the present teachings include methods of diagnosing or determining etiology of a pathogen- associated disease, in various configurations, these methods can comprise; a) providing at least one biological sample from a human subject; b) determining presence, absence and/or quantity of a bacterial pathogen, viral pathogen, or a combination thereof, by an assay selec ted from the group consisting of a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen D A detection test, a pathogen RNA detection test and a.
  • the present teachings include methods of diagnosing a viral pathogen-associated disease.
  • these methods can comprise: a.) providing at least one biological sample from a human subject; b) determining presence, absence and/or quantity of a virus selected from the group consisting of an adenovirus,, an enterovirus, HHV-6V or a combination thereof, by a pathogen culture, a serum, antibody detection test, a pathogen antigen detection test, a pathogen DMA detection test, a pathogen R A detection test, or a combination, thereof; c) determining in the sample, expression level of at least one endogenous gene that exhibits aberrant expression during infection with the virus, by an assay selected from the group consisting of a microarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain, reaction assay, a.
  • Southern blot assay a Northern blot assay, Western blot assay, an. enzyme-Unked immunosorbent assay and a combination thereof, whereby the subject, is diagnosed with the disease if the sample comprises a) the virus and b) an aberrant expression level of the at least one gene.
  • the present teachings include methods of diagnosing a
  • these methods can comprise: a) obtaining at least one biological sample from a human subject: and b) determining in the sample, expression levels of at least one endogenous gene selected from the group consisting of I I27, ISCi i 5, OTOF, M ' B, ITGAM, ITGAX and a combination thereof; by a microarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay, an enzyme-linked immunosorbent assay or a combination thereof, whereby the subject is diagnosed with the disease if the sample exhibits an aberrant level of expression of at least one gene selected from the group consisting of IFI27, iSGl 5, OTOF, IFIT3, ITGAM,
  • the disease can be Fever Withoui an Apparent Source.
  • the biological sample can comprise peripheral blood mononuclear cells.
  • the at least one aberrant level of expression can be at least a 2 fold increase or decrease compared to a control level, in various configurations of the embodiments, the at least one aberrant level of expression can be the at least one aberrant level of expression is at least a 1 ,5 fold increase or decrease compared to a control level.
  • the at least one gene associated wi th an adenoviral infection can be selected from the group consisting of ATM, PRKCH, PR CQ, C1QB, C!QC, IRF7, OAS3, QAS1 , OAS2, AK2, EIF2, 1FIH 1, DDX58, NOD2, TLR5,
  • NLRC4, TLR8, C3AR1 IL1 B, TLR1 , TLR4, TLR6, MYD88, CASP!, IF1T3, 1FI35, BCL2, MED 14, IFNG.R2, IFNARI , IRF1, BAX, IRF9, PSMB8, IFITMI , JA 2, STAT2, TAP1 , IKBKB, IKBKAP, K1AAI271, TRAP6, TNF, TB l, TANK, IRF9, NFKBIA, 1RF7, 1SGIS, ADAR.
  • ZB L il l 12 EOS, EY96, TLR5, IRA .3, TLR8, ⁇ 2 ⁇ 2, CD14, MAP J4, STAT4, HS.572649, CCR7, CD40LG, LIB, HLA-DOA, CREB5, FCGR3B, PCGR2A, IL1RN, LI ' BR, TYROBP, FCER.1G, FCGRiA, FCGR1 B and a combination thereof.
  • the at least one gene associated with a» adenoviral infection can be selected from the group consisting ofClQB, CIQC, 1RF7, OAS3, OA.S1, OAS2, AK2, EIF2, IF!H!, DDX58, NOD2, TLR5, ' LR.C4, TERS, C3AR1, IL!B, TLRL TLR4, TLR6, MYD88, CASPI, IFIT3, 1PF35, BCL2, IF ARI, IRFL BAX, 1RF , PSMB8, IFITM1, JAK2, STAT2, TAP.1, TRAF6, TNF, TBKl, ' FANE, IRF9,
  • NFKBIA NFKBIA
  • 1RF7 1RF7
  • ISG15 ADAR
  • ZBP1 IFIT2
  • FOS FOS
  • LY96 TLR5, .IRAK3, TLRS
  • EIF2A 2 CD 14, MAPK14, CREB5, FCGR3B, FCGR2A, IL1RN, LTBR, TYROBP, FCERIG, FCG IA, FCXI IB and a combination thereof, wherein the at least one gene has an aberrant level of expressio of at least a.1,5 fold increase compared to a control level
  • the at least one gene associated with an adenoviral infection can be selected from the group consisting of ATM, PRKCFL PRKLCQ, MED 14, IFNGR2, IK.BKB, IKBKAP, K1AA127I, STAT4, HS.572649, CCR7, CD40LG, LTB, HLA-DO.A and a combination thereof, wherein the at least one gene has an aberrant level of expression of at least a 1.5 ibid decrease compared to a control level.
  • the at least one gene associated with an enterovirai infection can be selected from, the group consisting of NLRC4, TLR8, TLR5, NOD2, KNASEL, TLR2, 1 ' LRl, C5A 1, MYD88, TLR6, CASPI, IL1B, CiQB, 1RF7, OASL OAS3, EIF2A 2, DDXS8, IFtFit, OAS2, !FITl, ⁇ 3, 1F135, MXL TAP I, STAT2, IFITM L STALL JAK2, IRF7, ISG1.5, 1FIT2, DHX58, ZBPL FOS, LY96, MAP2K3, MAPK14, CASP5 and a combination thereof
  • the at least one gene associated with an enterovirai infection can be selected from the group consisting of NLRC4, TLR8, TLR5, NOD2, RNASEL, TLR2, TJLRl, C5AR.1, MYD88, TLR.6, CASPI, IE IB, CIQB, IRF7, OASI, OAS3, B1F2A , DDX58, IFIHL OAS2, IFITL IFFF3, IFI35, M l, TAP 1, STAT2, IFITMJ, STATi, JAK2, IRF7, SSGI5, IF.IT2, DHX5S, ZBPL FOS, EY96, M P2K3, MAF I4, CASP5 and a combination thereof, wherein the at least one gene has an aberrant level of expression of at least a.1,5 fold increase compared to a control level,
  • the at least one gene associated with an BHV-6 infection can be selected from the group consisting of CCL5, PRKCH, TLR5, TLR2, NOD2, TLR8, TLR4, TLR6, C QA, MYD88, R1PK2, !LIB, C3AR1, CASP1, CTQB, CIQC, OASURF7, OAS3, EIF2A 2, TLR7, OAS2, DDX58, IFffl L IFIT3, IFITI, SOCSL MX1 , IFI35, IFITM1 , TAP!
  • the at least one gene associated with an BHV-6 infection can be selected from the group consisting of TLR5, TLR2, MOD2, TXR8, TLR.4, TLR.6, C.1.QA, YDS8, R.1PK2, [LIB, C3AR.1 , CASP1 , C1QB, CIQC, OAS1, 1RF7, OAS3, EIF2AK2, TLR7, OAS2, DDX58, IF!HL IFIT3, IFITI, SOCS l, MX1 JFI3S, IFSTMl, TAPL STAT2, JAK2, STAT.1 , TYR.OBP.
  • the at least one gene associated with an HHV-6 infection can be selected from the group consisting of CCL5, FRKCH and TLR9, wherein the at least one gene has an aberrant level of expression of ai least a 1.5 fold decrease compared to a control level.
  • the at least one gene can he selected from the group consisting of MYFI9, ARAP3, CD Ni C, MT2 , SPATS2L, IRF7, ZBPl , OAS2, OASL ISG15, IF16, HERC5, OAS3, RSAD2, OASL, IFIT3, OTOF and IFI27.
  • the at least one gene associated with a viral infection can be selected from the group consisting of CDK.N 1 C. MT2A, SPATS2L, IRF7, ZBPl , OAS2, OASI , LSG15, IF16, HERC5, OAS3, RSAD2, OASL, IP II 3, OTOF and IFI 27, wherein the at. least one gene has an aberrant level of expression of at l east a 1.5 fold increase compared to a bacterium-infected control level.
  • the at least one gene can be selected from the group consisting of MYH9 and ARA.P3, wherein the ai least one gene has an aberrant level of expression of at least a 1.5 fold decrease compared to a bacterium-infected control level
  • the at least one gene can be selected from the group consisting of BAK1 , R AS, ACTR2, NCK2, PIK3CB, MAP2K4, ITGA2B, ITGB3, MYLK, YL9, 1TGB5, GNG I 1 , ZYX, STGAX, IFNGRl , ITGAM, STAT2, IFI35, MX J , OASI , IFITI and SF1T3,
  • the at least one gene associated wi th a viral infectio can be selected from the group consisting of 8AKL IFNGRl, STAT2, IF135, MX I, OAS I , IFITI and - IFIT3, wherein the at least one gene has an aberrant level of
  • the at least one gene can be selected from the group consisting of RRAS, ACTR2, NCK2, PIK3CB, AP2K4, 1TGA2B, 1TGB3, MYLf MYL9, ITGB5, GNG 11. ZYX.
  • the at least one gene can be selected from, the group consisting of OSBPL8, VHL, ACTR2, MAP2 4, FBA 1, PRO i, ITOB3, MYL9, .1TGA2B, ITGB5, GNG 1 1 , EP300, ZYX, ARAP3, AGER, ITGAX, SORL1, IFNG l, IFNGR2, ITGAM, MT2A, SPATS2L, OAS2, OAS.l , 1SG15, IFI6, ⁇ , HERC5, OAS3, RSAD2, OASL OTOF and IFI27.
  • the at least one gene can be selected from the group consisting of lFNGRI , IFNGR2, MT2A, SPA.TS2L, OAS2, GAS1, ISG15, IFI6, 1FIT1 , HERC5, OAS3, RSAD2, OASL, OTOF and IFI27, wherein the at least one gene has an aberrant level of expression of at least a. 1.5 fold increase compared to a bacterium- infected control level.
  • the at least one gene can be selected fro.ra the group consisting of OSBPL8, VHL, ACTR2, MAP2K4, FI3AI, PROS!
  • the at least one gene has an aberrant level of expression of ai least a 1.5 fold decrease compared to a bacterium-infected control level.
  • the at least one gene can be selected from the group consisting of iFI ' 27, iSG!
  • the at least one gene can be selected from the group consisting of 1FI27, 1FIT3, and a combination thereof, whereb the subject can be diagnosed with febrile HHV-6 if the subject has an aberrant level of expression of at least a 1.5 fold increase of the at least one gene selected from the group consisting of IFI27 and.
  • IFFF3 compared, to an afebrile HHV-6 control level or a febrile control level
  • ihe ai least one gene can be selected from the group consisting of IPI27, ISG15, and a combination thereof, whereby the subject ca be diagnosed with, febrile adenovirus if the subject has an.
  • the at least one gene selected from the group consisting of IFI27 and ISO 15, compared to an afebrile adenovirus control level or a febrile control level in some configurations
  • the at least one gene can be selected from the group consisting of IF 127, ISO 15 and 1FIT3, whereby the subject is diagnosed with febri le enterovirus if the subject has an aberrant level of expression of at least a 1 ,5 fold increase of the at.
  • the at least one gene can be selected from the group consisting of IFI 27, ISO 15 and IFIT3, compared to an afebrile enterovirus control level or a febrile control level, hi some configurations, the at least one gene can be selected from the group consisting of ITGAM and ITGAX, whereby the subject can be diagnosed, with a bacteria.1 infection if the subject has an aberrant level of expression of at least a 1.5 fold increase of the at least one gene selected from the group consisting of ⁇ and TIG AX, compared to an afebrile control level or a viral-infected control level
  • diagnostic acc uracy can be at least 70%.
  • diagnostic accuracy can be at least 75%. in various configurations of the embodiments, diagnostic accuracy can be at. least. 80%, In various configurations of the embodiments, diagnostic accuracy can be at least 85%. In various configurations of the embodiments, diagnostic accuracy can be at least 90%.
  • the present teachings include methods of determining etiology of a disease, such as, without limitation . , Fever Without an Apparent Source (FWS) in a subject such as. for example, a human child.
  • these methods can include a) providing a peripheral blood sample from a human subject; b) determining presence, absence and/or quantity of a bacterial pathogen, a viral pathogen, or a combination thereof, by a pathoge culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DNA detection test, a pathogen RN A detection test, or a combination thereof; c) determining in the sample, expression levels of at least one endogenous gene in which aberrant expression levels are associated with infection with a pathogen, by a mieroarray hybridization assay, an RN A-seq assay, a polymerase chain, reaction assay, a LAMP assay, a ligase chain reaction assay,
  • the present teachings include therapies selected on the basis of a transcriptional profile determined by the disclosed methods. These can include, without limitation, selection and administratio of an appropriate antibiotic in the. case of a disease determined to he caused by a bacterial infection, or selection, and administration of an appropriate anti-viral drug in the case of a disease determined to be caused by a viral infection.
  • the present teachings include an antibiotic for use in a method of treatment of Fever Without an Apparent Source (FWS) in a subject, characterized
  • the subject has in a biological sample such as a peripheral blood sample a least a .1 ,5 fold increase in expression level compared to a control level of at least one endogenous gene, such as, without limitation, at leas one endogenous gene selected from the grou consisting FYR, CD247, EITPR3, CD3, ZAP7Q, PLCG1 , PRKCH, LC , LA I; PRKCQ, ⁇ , RHOU, G A13, PPPl R12A. RHOTI, FCER.lG, LYN, RALB, GNAQ, MARC S, TGM2,
  • the present teachings include an antibiotic for use in a method of treatment of a disease caused by bacteria, characterized in that the patient has at least a 1 ,5 fold increase in transcription levels of one or more genes that increase in expression level upon bacterial infection, and/or a t least 1 , 5 fold decrease in transcription levels of one or more genes that decrease in expression level upon bacterial infection.
  • the present teachings include an anti-viral drug for use m a method of treatment of a disease caused by a virus, characterized in that the patient has at least a L5 fold, increase in transcription levels of one or more genes that increase in expression level upon viral infection, and/or at least a 1.5 fold decrease in transcription levels of one or more genes that decrease in expression level upon viral infection.
  • the present teachings include an antibiotic for use in a method of treatment of a disease caused by bacteria, characterized in that the patient has been selected to have at least, a i .5 fold increase in transcription levels of one or more genes that increase in expression level upon bacterial infection, and/or at least a 1.5 fold decrease in transcription levels of one or more genes that decrease in expression level upon bacterial infection.
  • the present teaching include an anti-viral drug for use in a method of treatment of a. disease caused by a virus, characterized i that the patient has been selected to have at least at least a 1.5 fold increase in transcription levels of one or more genes that increase in expressio level upon viral infection, and/or at least, a 1.5 fold decrease in transcription levels of one or more genes that decrease in expression level upon viral infection.
  • the present teachings include an antibiotic for use in a method of treatment of a disease caused by bacteria, characterised in that the method comprises a determination, of whether the patient has at least a 1.5 fold increase in transcription, levels of one or more genes that increase in. expression level upon bacterial infection, and/or at. least a 1.5 fold decrease in transcription levels of one or more genes that decrease in expression level upo bacterial infection.
  • the present teachings include an. anti-viral drug for use in a method of treatment of a disease caused by a virus, characterized in that the method comprises a determination of whether the patient has at least a L5 fold increase in
  • transcription levels of one or more genes that increase in expression level upon viral infection and/or a t least a. 1.5 fold decrease in transcription levels of one or more genes that decrease in expression level, upon viral infection.
  • the present, teachings include an antibiotic for use in a method of treatment of Fever without an Apparent Source (FWS) in a subject, characterized in that the subject has in a sample such as a blood sample at least, a 1.5 fold increase in expression level compared to a control level of at least one endogenous gene selected from the group consisting of RHOU, GNAI 3, PPP1 S 2A, RHOT1, FCER1G, LYN, RALB, GNAQ, MARCKS, TGM2, ARHGEF1 1 , MYL12A, EP300, MYL9, CREB5, FCGR2A, GNG10, GNGI 1, C1QR NOD2, TLR2, TLRi , R.NASEL, C5A.RE TLR4, MYD88, P1 3CB, C ARE TLR0.
  • a sample such as a blood sample at least, a 1.5 fold increase in expression level compared to a control level of at least one endogenous gene selected from the group consisting of
  • NERC4, TER8. ILIB, PPP1R.E ZYX, YL12 , VA.SP, UGA2B, ITGB5, VCL, 1TGB3, M YLK, ASAPE ITGAM, ITGAX.
  • the present, teachings include an antibiotic for use in a method of treatment of Fever without an Apparent.
  • Source (FWS) in a subject characterized in that the subject has in a sample such as a blood sample at least a .1.5 fold increase in expression ievel compared to a control level of at least one endogenous gene selected from the group consisting of FY , CD247, EITPR3, CD3, ZAF70, PLCG L PRK.CFL LCK, EAT, PRK.CQ, ⁇ , ITGB7, TSPAN4, KXRDE IR2DE3, KIR2DE4, K1R3DL3, IR3DLE HCST, C.D247, NCR3 and a combinatio thereof.
  • the present teachings include an antibiotic for use in a method of treatment of Fever without an Apparent Source (FWS) in a subject, characterized in that the subject has in a blood sample at least a 1.5 fold decrease in expression level compared to a control level of Annexin A3 gene.
  • the present teachings include an anti-viral drug for use in a method, of treaiment of Fever with an Apparent Source (FWS) in a subject.
  • the F WS can be characterized in thai the subject can have in a sample such as a blood sample at least a 1.5 ibid increase in. expression level compared to a control level of at least one endogenous gene selected from the group consisting of IF 127, ISO 15, Ol OF, IF1T3, ITGAM, ITGAX and a combination thereof.
  • the present teachings include an anti-viral drug for use in a method of treatment of an adenoviral infection in a subject, characterized in that, the subject can have in a sample such as a blood sam le an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of ATM., PRKCFL PRKCQ, C IQB, CI QC, IRF7, OAS3, OAS1 , OAS2, AK2, ⁇ 2, IPIHl , DDX58, NOD2, T.LR5, NLRC4, TLR.8, C3AR.1 , ILI B, TLR1 , TLR4, TLR.6, MYD88, CASP!, IFIT3, 1FI35 BCL2, MED 14.
  • a sample such as a blood sam le an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of ATM
  • the present teachings include an anti-viral drug for use in a method of treatment of an adenoviral infection in subject, characterized In that the subject can hav e in a sample such as a blood sample an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting ofClQB, CIQC, IRF7, OAS3, OAS I, OAS2, AK2, EIF2, IFIHE DDX58, NOD2, TLR5, NLRC4, TLR8, C3ARL ILI B, TLR l , TLR.4, TLR.6, MYD88, CASPL IFIT3, IFI35, BCL2, IFNARI, !RFL BAX, 1RF9, PS B8, IFITMi , JAK2, STAT2, TAPE TRAF6, TNF, TB3K K TANK, IRF9, NFKBIA, IRF7, 1SG15 ADAR, ZBP l
  • FCGR2A ILI N, LTBR, TYROBP, FCERIG, FCGRI A, FCGRIB and a combination thereof.
  • the present teachings include an anti-viral drug for use in a method of tTeatraeni of an adenoviral infection i a subject, characterized in that the subject can have in. a sample such as a blood sample an aberrant level of expression, at least a 1 ,5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of ATM, PRKCFL PRKCQ, MED 14, IFNGR2, IKBKB. IKBKAP, IAA127 L STAT4, HS.572649, CCR7, CD40LG, LT.B, HLA-DOA and a combination thereof and a combination thereof,
  • the present teachings include an anti-viral drug for use in a method of treatment of an enteroviral infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant level of expression at least a 1 , 5 fold greater compared to a control level of at least one endogenous gene selected from the group consisting of NL C4, TLR8, TLR5, NOD2, RNASBL, TLR2, TLRl, CS RI , YD88, TLR6, CASP1, IL1B, C iQB, IRF7, OAS1, OAS 3, BFF2AK2, DDX58, IFIM, OAS2, If IT!, IFST3, IFB5, M 1 , TAF1, STAT2, IFITM1 , STAT I , JAK2, IRF7, ISG15, T2, DMX58, ZBP1, FOS, LY96, MAP2K3, MAPKI4, CASP5 and a combination thereof
  • the present teachings include an anti-viral drug for use in a method of treatment of an enteroviral infection in a subject, characterized in that the subject can have in a sample such as a. blood sample an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting ofCCLS, PR CH, TLR5, TLR2, NOD2, TLR8, TLR4, T ' LR6, CI OA, MYD88, RIPK2, IL! B, C3AR.1, CASPL CI QB.
  • CI QC OAS 1. 1RF7, OAS3, EIF2AK2, TLR7, OAS2, DDX58, IFIHL IPIT3, l.FLT ' S , SOCSI , MX ' L FB5, IFIT L TAPL STAT2, JAK2, STAT1 , TYROBP, CD86, IL1B, CA.SP5, CCL2, TLR9, TRA.F6, IRAK4, CHUI , CD 1.4, MYD88, TLRl , FOS, LY 6 and a combination thereof.
  • the present teachings include an anti-viral drug for use in a method of treatment of an enteroviral intection in a subject, characterized in that the subject can ha ve in a sample such as a blood sample an aberrant level of expression, at least a 1.5 fold greater compared to a control level of at least one endogenous gene selected from the group consisting of NLRC4, TLR8, TLR5, MOD2, RNASEL, TLR2, TLRl , C5.AR1 , MYD88, TLR.6, CASPL ILI B, C1QB, IRF7, OAS 1 , OAS 3, E1F2AK2, DDX58, IF 11:11, OA.S2, ⁇ , 1P3T3, IF135, MX1, TAPL STAT2, IFITML STAT L JA 2, IRF7, ISO 15, IFIT2, DFIX58, ZBPf, FOS, LY96 " , MAP2K3, MAPK14, CASP5 and a combination thereof
  • the present teachings include an anti-viral drug for use in a method of treatment of an enteroviral infection in a subject, characterized, in that the subject has in a blood sample an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of CCL5, PRKCH, TLR5, TLR2, NOD2, TLRB, TLR4, TLR6, C1QA, MYD8S, RIPR2, ILI B, C3ARL CASPL CIQB, CiQC, OAS I, IRF7, OAS3, EIF2AK.2, TLR7, OAS2, DDX58, IFffl L ⁇ 3, IFIT1 , SOCSI , M.X 1 , IFD5, IFITML TAP STAT2, IAK2, STAT1 , TY.ROBP, CD86, IL1 B ; CASP5, CC.LL TLR9, TRAFi IRA 4, CHUK, CD14, MY
  • the present teachings include an anti-viral drug for use i a method of treatmen t of an ' HHV-6 infection in a subject, characterized in thai the subject can have in a sample such as a blood sample an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting ojf CCL5, FRKCH, TLR9 and a combination thereof
  • the present teachings include an anti-viral drug for use hi a method of treatment of an BHV-6 infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant level of expression of at least 1 ,5 fold increase compared, to a control level of al least one endogenous gene selected from, the group consisting ofCD N!C MT2A, SPATS2L, 1RF7, ZBP1 , OA .2, OAS 1, 1SG1 S, IF.16, HERC5, OAS3, RSAD2, OAS!,, IF1T3, OTOF and IF 27.
  • a control level of al least one endogenous gene selected from, the group consisting ofCD N!C MT2A, SPATS2L, 1RF7, ZBP1 , OA .2, OAS 1, 1SG1 S, IF.16, HERC5, OAS3, RSAD2, OAS!,, IF1T3, OTOF and IF 27.
  • the present teachings include an anti-viral drug for use in a method of treatment of an. HHV-6 infection in a subject, characterized in that the subject can have in a sample such, as a blood sample an aberrant level of expression of at least 1 .5 fold decrease compared to a control level of at least one endogenous gene selected from the group consisting of YH9 and ARAP3.
  • the present teachings include an anti-viral drug ' for use in a method, of treatment of an HHV-6 infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant le vel of expression of at least 1.5 fold increase compared to a. control level of at least one endogenous gene selected irom the group consisting of CD N1C, MT2A, SPATS2L, IRF7, ZBPL OAS2, OAS1 , ISG15, TFI6,
  • the present teachings include an antibiotic for use in a method of treatment of a bacterial infection in a subject, characterized in that a sample such as blood sample of the subject can. have at least a 1 ,5 fold decrease in expression, of at least one endogenous gene selected from the group consisting of CD N1C, MT2A, SPATS2L, IRF7, ZBPI , OAS2, OAS I, 1SG15, FJ.6, HERC5, OAS3, RSAD2, OASL 1FIT3, OTOF and FI27.
  • the present teachings include an antibiotic for use in a method of trea tment of a.
  • bacterial infection in a subject characterized in thai a sample such as a blood sample of the subject can have at least a 1.5 fold increase in expression of at least one endogenous gene selected from the group consisting of MYH9, ARAP3 o a combination thereof.
  • the present teachings include an antibiotic for use in a method of treatment of a bacterial infection in a subject, characterized in that a sample such a a blood sample of the subjec t can have at least a 1.5 fold decrease in expression of at least one endogenous gene selected from the group consisting of CD NiC, MT2A, SPATS2L, I F7, ZBPL OAS2, OAS I, ISO 15, 1-16, MERCS, OAS3, RSAD2, OASL, lFil ' 3, OTOF a d 27, and at least 1.5 fold increase in expression of at least one endogenous gene selected from the group consisting of YB.9, ARAP3 and a combination thereof.
  • the present teachings include an antibiotic for use in a method of treatment of a bacterial infection, characterized, in that, a sample such as a. blood sample of the subject can have an aberrant level of expression at least 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group
  • the present teachings include an antibiotic for use in a method of treatment of a bacterial infection, characterized in that a sample such as a blood sample of the subject can have an aberrant level of expression that is at least a F5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group
  • the present teachings include an anti-viral drug for use in a method of treatment of an enieroviral infection in a subject, characterized, in that the subject can have in a sample such as a blood sample an aberrant level of expression that is at least, a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of adenoviral, infection is selected from the group consisting of NERC4, TER8, TLR5, OD2, RNASEL, TLR2, TLRl , C5AR1, MYD88, TER6, CASPE IL1B, CI QB, IRF7, OASE OAS3, E1F2AK2, DDX58.
  • the present teachings include an anti-viral dru for use in a method of treatment of an enteroviral infection in a subject, characterized in that the subject can have in a sample such as a blood sample -an aberrant level of expression that is at least a 1,5 fold increase compared to a control level of at least one endogenous gene selected from the group consisting of NERC4.
  • TER8. TLR5. NOD2, RNASEL, TLR2, TLRl , C5AR1 , MYD88, TLR6, CASPE I LI B, CIQB, IRF7, OAS I, OAS3, EIF2AK2, DDX5S, IFIH E OAS2, IF1TL 1FIT3, IFI35.
  • the present teachings include an anti-viral drug for use in a method of treatment of an HBV- infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant, level of expression that is at least a 1.5 fold decrease compared to a control level of at least one endogenous aene selected from the group consisting of CC1.5, PR CH and. TLR9.
  • the present teachings include an anti-viral drug for use in a meth od of treatment of a viral infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant level of expression that is at least a 1.5 fold increase of at least one endogenous gene selected from the group consisting of CDKNIC, MT2A, SPATS2L, IRF7, ZBPE OAS2, OAS E ISGI5, IPI6, HERC5, OAS3, RSAD2, OASE, IFFT3, OTOF and IFI27, compared to a bacterium-infected control level.
  • a sample such as a blood sample
  • an aberrant level of expression that is at least a 1.5 fold increase of at least one endogenous gene selected from the group consisting of CDKNIC, MT2A, SPATS2L, IRF7, ZBPE OAS2, OAS E ISGI5, IPI6, HERC5, OAS3, RSAD2, OASE, IFFT3, OTOF and I
  • the present, teachings include an anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in. that the subject has in a sample such as a blood sample an aberrant level of expression that is at. least, a E5 fold decrease of at least one endogenous gene selected from the group consisting of MYH9 and ARAP3, compared to a bacterium-infected control level.
  • the present teachings include an anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in thai the subject can have in a sample such as a blood sample an aberrant level of expression that i at least a 1 ,5 fold increase of at least one endogenous gene selected from the group consisting of BAK E 1FNGRI , STAT2, IF135, MX I, OAS! , IFIT1 and IFIT3, compared to a bacterium-infected cont.ro! level.
  • the present teachings include an anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in. that the subject can have in a sample such, as a blood sample a aberrant level of expression thai is at least a 1.5 fold decrease of at least one endogenous gene selected from the group consisting of RRAS, ACTR2, CK2, PDC3CB, MAP2K4, ⁇ .2 ⁇ , ITGB3, MYLK, MYL9, II ' GBS, GNG 1 L ZYX, ITGAX and ITGAM, compared to a bacteriura-infected control level
  • the present teachings include an anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant level of expression that is at least a 1.5 fold increase of at least one endogenous gene selected from the group consisting of IFNGRl , IFNGR2, MT2A, SPATS2L, OAS2, OAS I, iSG l S, IFI6, iFITL HERC5, OAS3, RSAD2, OASL, OTOF and 1FI27, compared to a bacterium-infected control level.
  • a sample such as a blood sample
  • an aberrant level of expression that is at least a 1.5 fold increase of at least one endogenous gene selected from the group consisting of IFNGRl , IFNGR2, MT2A, SPATS2L, OAS2, OAS I, iSG l S, IFI6, iFITL HERC5, OAS3, RSAD2, OASL
  • the present teachings include an anti-viral drug for use in a method of trea tment of a viral infection in a subject, characterized in that the subject can have in a blood sample an aberrant level of expression that is at least a 1 .5 fold decrease of at least one endogenous gene selected from the group consisting of OSBPL8, VHL, ACTR2, MAP2K4, P13A1 , PROS1 , ITGB3, MYL9, ITGA2B, !TGBS, GNG1 1 , EP300, ZYX, ARAP3.
  • AGER, ITGAX, SORLI and KJTAM compared to a bacteriura-infected control .level.
  • the present teachings include an anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant level of expression that is at least a 1.5 fold- increase of at least one endogenous gene selected from the group consisting of 1FI27, ISO 15, OTOF and IFIT3, compared to a bacterium-infected control level
  • the present teachings include an anti-viral, drug for use hi a method of treatment of febrile HHV-6 in a subject, characterized i that the subject can be selected to have in a sample such, as a blood sample expression that is at least a .1.5 fold increase of at least one endogenous gene selected from the group consisting of IF127, IFi ' O, and a combination thereof compared to an afebrile HHV-6 control level or a febrile control level.
  • the present teachings include an anti-viral drug for use in a method of treatment of febrile HHV-6 in a subject, characterized in that the subject can be selected to have in. a sample such as a blood sample expression that is at least a IS fold increase of at least one endogenous gene selected from the group consisting of IF127, JSCS 15, and a combination thereof compared to an afebrile adenovirus control level or a febrile control level
  • the present teachings include an anti-viral drug for use in a meihod of treatment of febrile enterovirus in subject, characterized in that the subject can be selected to have in a sample such as a blood sample expression that is at least a 1.5 fold increase of at least one endogenous gene selected Irom the group consisting of IFI27, ISO 15, and a combination thereof, compared to an afebrile enterovirus control level, or a febrile control level.
  • the present teachings include an. antibiotic for use in a method of treatment of bacterial infection in a subject, characterized in that the subject can be selected to have in a blood sample expression tha is at least a 1.5 fold increase of at least one endogenou gene selected from the group consisting of ITGAM and ITGAX, compared to an afebrile control level or a viral-infected control level,
  • FIG. 1 illustrates identificationio of virus- and bacterial response-specific probes
  • FIG. 1 A illustrates Venn diagram showing identification of virus- and bacterial response-specific probes
  • FIG. I B illustrates heat maps of gene expression of viral response-specific probes
  • FIG. IC illustrates heat maps of gene expression, of bacterial response-specific probes.
  • Each row represents a gene with expression value normalized to the mean of the afebrile virus- negative control group and each column represents one individual, in original color, red represents up-regulation and blue represents down-regulation.
  • FIG. 2 illustrates blood leukocyte transcriptional profiles of febrile and afebrile HFiV-6- positive children compared t those of afebrile virus-negative children
  • FIG. 2A illustrates a clustering of probe sets.
  • FIG. 2B illustrates principal component analysis of differentially expressed genes, with each oval representing one child.
  • FIG. 2C, FIG 2D, FIG. 21-3, and FIG. 2F illustrate clustering of differentially expressed genes of FIG. 2A according to expression intensity In ingenuity® canonical pathways.
  • Each row represents a gene whose expression value is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left.
  • Each column represents one individual. In original color, red represents up- regulation and blue represents down -regulation.
  • FIG, 3 illustrates probes specific for individual viruses and for bacteria.
  • Each .row represents a probe and each column displays probes for one febrile subject positive for the indicated virus or with acute bacterial infection.
  • FIG- 4 illustrates classifier probes discriminating febrile children positive ibr viruses from febril children with acute bacterial infections
  • FIG, 4A illustrates 1581. gene-based classifiers
  • FIG. 4B illustrates 18 gene-based, classifiers
  • FIG, 4C illustrates 22 pathway-based classifiers
  • FIG. 41) illustrates 33 classifiers selected from, gene-based and pathway-based classifier sets.
  • Class is designated by green (viral) or blue (bacterial) letters: A, adenovirus; B. Bacteria; E, enterovirus; II, HH V-6, Classification based on patients * white blood cell ( WBC) count is shown beneath each heat map.
  • the upper strip shows classification based on age-specific normal values and the lower strip shows classification based on a cutoff of 15,000 cells per cii mm.
  • FIG. 5 illustrates blood transcriptional profiles of febrile adenovirus-positive children and profiles of afebrile adenovirus-positive children and afebrile controls.
  • FIG. 5A illustrates clustering of probe sets.
  • FIG, SB illustrates a principal component, analysis of differentially expressed genes, with each oval representing one child.
  • FIG. SC. FIG. 51), FIG. 5E, FIG. 51% and FIG. 5G illustrate clustering of differentially expressed genes from FIG . 5 A according to expression intensity in Ingenuity® canonical pathways.
  • Each row represents a gene with expression value that is normalized to the mean of the afebrile virus-negative- control group. Gene names are listed to the left.
  • Each column represents one individual. In original color, red represents up-regulation, and blue represents down-regulation.
  • FIG, 6 illustrates blood transcriptional profiles of enterovirus-positive febrile children and virus-negative afebrile children.
  • FIG. 6 A illustrates clustering of probe sets.
  • FIG. 6B illustrates a principal component analysts of differentially expressed genes, with each oval representing one child, FIG. 6C, FIG 61), FIG. 6 ⁇ , FIG. 6F, and FI.G. 6G illustrate clustering of differentially expressed genes in FIG. 6A according to expression intensity in Ingenuity® canonical pathways-
  • Each row represents a gene with expression value that is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left.
  • Each column represents one individual. In original color, red represents up-reguHuiou, and blue represents down-regulation,
  • FIG. 7 illustrates blood transcriptional profiles of febrile children with acute bacterial infections and profiles of virus negative afebrile children.
  • FICi. 7A illustrates clustering of probe sets.
  • FIG, 7B illustrates a principal component analysis of differentially expressed genes, with each oval representing one child.
  • FIG. 7C, FIG 7D, FIG. 7E, FIG. 7F, and FIG. 7G llustrate clustering of differentially expressed genes in FIG. 7 A according to expression intensity in ingenuity® canonical pathways.
  • Bach row represents a gene with expression value that is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column represents one individual. In original color, red represents up-regulation, and blue represents down-regulation.
  • FIG. 8A and FIG. 8.8 illustrate selected up- and down-regulated Ingemnty 5 canonical pathways identified for febrile children positive for adenovirus, HHV-6, or enterovirus and febrile children with acute bacterial infections.
  • the pathways are arranged, in ascending order by average P value of four infections for a pathway.
  • FIG. 9 illustrates quantile -normalized raw signal intensity of the classifier probes in febrile children.
  • FIG. 9A illustrates 18 classifiers identified from. 260 viral- and 1 ,321 bacterial response-specific probes.
  • FIG. 9B illustrates 22 classifiers identified from 34 genes in the Ingenuity® IFN signaling pathway and 205 genes in the Ingenuity® integral signaling pathway.
  • FIG. C illustrates 33 classifiers identified from using both gene-level and pathway- based approaches.
  • FIG. 9D illustrates relative expression data of nine classifier genes. In FIG. 9 A - 9D, each dot represents one sample.
  • FIG. 10 illust.ra.tes validation of three sets of classifier probes discriminating virus-positive febrile children from febrile children with acute bacterial infection using three independent cohorts of subjects
  • FIG . 10A illustrates validation with a set of 785 probes overlapped across three datasets with 1 ,58.1 virus- and bacterial response-specific probes.
  • FIG. 10B illustrates validation with gene-based classifiers.
  • FIG. IOC illustrates validation with pathway-based classifiers
  • FIG. iOD illustrates validation with hybrid gene- and pathway-based classifiers.
  • FIG. 10A ⁇ FIG. 10D Patient groups are indicated by stripes at the top of the heat map, hi original color, predicted class is labeled with green for viral or blue for bacterial infection.
  • Heat map rows are gene probes, whereas columns are individual subjects.
  • FIG. 1 1 illustrates samples from febrile subjects with confirmed viral/bacterial infection.
  • FIG, ⁇ 1 A illustrates correlation of transcriptional changes and leukocyte subpopulations in the subjects
  • FIG, 1 IB illustrates an expression, pattern of corresponding 4,716 probe sets in cluster format. Rows represent genes and columns represent individual samples, Dashed lines indicate the lowest, values of correlation coefficients significant (adjusted P ⁇ 0.05) for each parameter.
  • RNA-seq RN A Sequencing, Whole Transcriptome Shotgun Sequencing
  • microarray refers to an ordered arrangement of hybridkab!e array elements including but not limited to polynucleotide probes, on a substrate.
  • biological sample is any sampling of cells, tissues, or bodily fluids containing cells.
  • biological samples include, but are not limited to, peripheral blood mononuclear cells, a nasopharyngeal, sample, a urine sample, a blood sample, a lumbar puncture sample, a bodily fluid, a biopsy sample, a tissue sample and a combination thereof,
  • blood ncludes whole blood, plasma, and serum.
  • allelic expression levels includes an increase or decrease of gene expression compared to baseline, or compared to a appropriate comparison group.
  • Methods of gene expression profiling can include methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides.
  • Methods for quantification of niRNA expression in a sample include northern blottin and in situ hyb idization (Parker & Barnes, Methods in Molecular Biology 106:247-283 ( 1.999)); RNAse protection assays (Hod, Y., Biotechniques 1 92, 1.3:852-854); and. reverse transcription- polymerase chain reaction (RT-PCR) (Weis et at., Trends in Genetics 8:263-264 (1992)).
  • RT-PCR reverse transcription- polymerase chain reaction
  • antibodies can be employed that can recognize specific duplexes, including DMA duplexes, RNA duplexes, and DNA-R . hybrid duplexes or O A-protein duplexes.
  • Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSSK For example, RT-PCR can be used to compare mRNA levels in different sample populations, in normal and infected tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between, closely related niRNAs, and or to analyze RNA structure,
  • A, first step for an RT-PCR. analysis can be extraction and/or isolation of mRNA from a sample.
  • starting materia! can be total RNA isolated from a human PBMC.
  • RNA can be isolated from a variety of PBMCs, such as, without limitation, a lymphocyte, a leukocyte, a monocyte or a macrophage.
  • RNA isolation can be performed using a purification kit, buffer set and proteas from commercial manufacturers, such as Qiagen, according to manufacturer's instructions. For example, total RNA from cells in culture can be isolated usina Qia3 ⁇ 4en RNeasv® mini-columns. Other commercially available RNA isolation kits include MasterPureTM Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), and Paraffin Block RNA Isolation Kit (Ambion, inc.). RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test), RNA prepared from a biological sample can be isolated, for example, by cesium chloride densit gradient cenirifiigation.
  • A. first step in gene expression profiling by RT-PCR can be reverse transcription of an RNA template into cDNA, followed, by amplification in a PGR reaction.
  • extracted RNA can. be reverse-transcribed using a GeneAmp RNA PGR kit (Perkin Elmer, Walthani, MA USA), following manufacturer's instructions.
  • cDNA can then be used as template in a subsequent PGR amplification and quantitative analysis using, for example, a TaqMao® (Life Technologies, Inc., Grand island, NY) assay.
  • TaqMan.® RT-PCR (life Technologies, Carlsbad, CA, USA) can be performed using commercially available equipment, such as, for example, an ⁇ . ⁇ PRISM 7700TM Sequence Detection SystemTM (Perkiti-Elmer-Applied Biosystems, Foster City, Calif, USA), or Jighteye!er (Roche Molecular BioehemicaLs, Mannheim, Germany), RT-PCR can be performed using an internal standard such as mRNA for glyceraldehyde-3-phosphate- dehydrogenase (GAPDH) and/or ⁇ ---aetin as a control (see, e.g.. Held et Genome
  • GPDH glyceraldehyde-3-phosphate- dehydrogenase
  • PCR primers and probes can be designed based upon intron sequences present in the gene to be amplified, in such aspects, a first step in primer/probe design can be the delineation of intron sequences within, the genes. This can be accomplished using publicly available software, such as the DMA BLAST software (Kent, W. I. Genome Res, 12(4): 656-664, 2002). Subsequent steps can include the following established methods of PCR primer and probe design.
  • repetitive sequences within nitrons can be masked when, designing primers and probes. This can be accomplished by using software such as the Repeat Masker program available on-line through the Baylor College ofMedicine, This, program can be used to screen DNA sequences against a library of repetitive elements and returns a. query sequence i which the repetitive elements are masked.
  • Masked intron sequences can then be used to design primer and probe sequences using a commercially or otherwise publicly available primer/probe design package, such as Primer Express (Applied Bsosysteras); MGB assay-by-deslgn (Applied Biosystems); Pomer3 (Rozen, and Skaietsky, H.J., (2000) Primer3 on the WWW for general users and for biologist programmers.
  • Primer Express Applied Bsosysteras
  • MGB assay-by-deslgn Applied Biosystems
  • Pomer3 Razen, and Skaietsky, H.J., (2000) Primer3 on the WWW for general users and for biologist programmers.
  • Factors considered in PCR primer design can include primer length, melting temperature (Tra), G/C content, specificity, complementary primer sequences, and 3 -end sequence- PCR primers can be, but are not limited to, 1.7-30 bases in length, and contain about 20% from 0% to 80%, or about 80% G - C bases, such as, for example, about 50%, from 50 to 60%, or about 60% G+C bases, in various configurations, Tm's can be between 50 and 80° C Fundamental e.g. about 50 to 70° C.
  • PCR primer and probe design can be found in various published sources, e.g. Dieffenbach, C. W. et L, "General Concepts for PCR Primer Design” in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 533-3.55; innls and Geifaod, "Optimization. of PCRs” in: PCR Protocols, A Guide to Methods and Applications * C C Press, London, 1994, pp. 5-1 1 ; and Plasterer, T. N, Priroerselect: Primer and probe design. Methods Mol. Biol. 70:520-527 ( 1 97), the entire disclosures of which are hereby expressl y incorporated by reference.
  • differential gene expression can be identified, or confirmed usi ng a microarray technique.
  • polynucleotide sequences of interest including cDNAs and oligonucleotides
  • the arrayed sequences can be hybridized with specific DNA probes from cells or tissues of interest.
  • RNA can be isolated from a variety of biological sources.
  • PCR-amplified inserts of cDNA clones can be applied to a substrate in a dense array. Mieroarrayed genes, immobilized on a microchip, can be suitable for hybridization under stringent conditions.
  • fluorescently labeled cDNA probes can be generated through incorporation of fluorescent nucleotides by reverse transcription: of RNA extracted from tissues of interest
  • Labeled cDNA probes applied to a chip can. hybridize with, specificity to loci of DNA on the array. Afte washing to remove non-specificaily bound probes, a chip can be scanned by confocal laser microscopy or by another detection method, such as a CCD (charge coupled device) camera. Quantification of hybridization of each arrayed element, can allow for assessment of corresponding mRNA.
  • CCD charge coupled device
  • dual color fluorescence can be used.
  • dual color fluorescence separately labeled cDNA probes can be generated from two sources of RNA and can be hybridized pairwise to an array. The relative abundance of transcripts from the two sources corresponding to each specified gene can be determined simultaneously.
  • the miniaturized scale of the hybridization can afford a convenient and rapid evaluation of an expression pattern for large numbers of genes, in various configurations, such methods can have sensitivity to detect rare transcripts, which are expressed at fewer than 1000, fewer than 100, or fewer than 10 copies per cell, hi various configurations, such methods can detect at least approximately two-fold differences in expression, levels (Schena ei al., Froc. Natl. Acad. Sci.
  • microarray analysis can. be performed by commercially available equipment, following manufacturer's protocols, -such as by using AfTymetrk GenChip technology, or Ineyte's microarray technology.
  • Non-limiting representative protocols for profiling gene expression including mRNA isolation, purification, primer extension and amplification are given in various published journal articles (for example; Godf ey, T.E., t tL 2000, J, Molec, Diagnostics 2: 84-91 ; Spech.t, K, ei « ., 2001 , Am. J. Pathol. 1.58: 41.9-29),.
  • RNA can be extracted, and protein, and DNA can be removed..
  • Alter analysis of the RNA concentration, RNA repair and/or amplification steps can be included, and RNA can be reverse transcribed using gene specific promoters followed by RT-PG1. Data can be analyzed to identify a treatment option(s) available to the patient on th basis of the characteristic gene expression pattern identified in the examined biological sample.
  • Subjects were drawn from a study of children between 2 to 36 months of age with Fever Without an Apparent Source ( Table 1 ) and afebrile children having ambulatory surgery who were recruited at St Louis Children's Hospital as described previously (Colvin, J.M., et at. 2012, Pediatrics 1.30(6):e 1455- 1462).
  • the febrile and afebrile groups were similar with respect to age, gender, and season of recruitment, but differed with respect to race, with more African-American children in the " febrile group (57% vs. 13%).
  • Patients were enrolled according to Institutional Re view Board- ppr ved protocol. The study was approved by the Washington University Human Research Protection Office.
  • Each subject was tested for m ultiple viruses in blood and nasopharyngeal samples using panels of virus-specific PC assay as described (Colvin, J.M., el al. 2012, Pediatrics 130(6):el455-1462), Subjects were selected for the study of gene expression profiles if they were positive for a single virus in one or both samples.
  • the viruses included, were adenovirus, HHV-6, enterovirus, and rkinoviras.
  • This example illustrates preparation of biological samples, including RNA from blood samples.
  • RNA was isolated from whole blood collected in. TempusTM Blood RNA tubes (Applied Biosystems, Carlsbad, CA) according to the manufacturer ⁇ instructions. RNA quality was determined by gel-chip image (showing 28S, 18S and 5S bands) and RNA integrity number (RIN, generally a >? RIN indicates good quality RNA) using an Agilent 21.00 Bioanalyzer (Agilent, Palo A l to, CA), All but 3 of the RN A preparations had RIN scores > 7.0. Total R A concentration wa obtained from an absorbance ratio at 260.nm and 280nm using a NanoDrop ND-100 spectrometry instrument (NanoDrop inc., Wilmington, DE).
  • This example illustrates gene expression micfoarray assays.
  • RNA transcripts were amplified by T7 linear
  • Hybridization Assay Kit (Illumina inc., San Diego, CA)
  • biotin-labeled cRNA targets were hybridized to the Illumina Human-HT12 v4 Expression BeadChips (>47,000 probes), which were scanned on an Illumina BeadArray Reader. Scanned images were quaniitated by ' illumina Beadscan software (v3). Quantified data were imported into illumina GenomeStudio software (versio 201. 1.1) to generate expression profiles and to make data quality assessments. These data have been deposited into GEO database at the National Center for Biotechnology Information (GEO ID:
  • This example illustrates microarray data analysis including differential expression analysis, pathway analysis, and identification of classifier genes, class prediction and unsupervised hierarchical, clustering.
  • host transcriptional response was analyzed at the level of up- and down-regulation of individual genes and of functional gene pathways. Differences were detected of up - and down -regulation of individual genes in transcriptional profiles in febrile children positive for any of the three viruses and with acute bacterial infection.
  • Several approaches to developing panels of probes were used. A panel of individual gene probes was developed based on the strength of statistical association with a type of infection.
  • a panel was developed of genes from two pathways that differentially activated: the Interferon Signaling Pathway, activated in febrile virus-positive children, and the integrisi Signaling Pathway, activated in children with, acute bacterial infection,
  • a hybrid approach was additionally used, in which genes were selected, from each of the gene-based and pathway- based approaches.
  • pathways that were roost activated for each virus and for bacteria! infection were identified from the mgem»ty# Pathway Analysis (Ingenuity® Systems, Redwood City, CA) library of canonical pathways.
  • the significance of the association between the data set and the canonical pathway was assessed in. two ways: 1 ) The ratio of the number of up- and down-regulated probes from the da ta set included in the pathway divided by the total number of probes that were included in the canonical pathway; 2) statistical evaluation using Fisher's exact test of the probability that the association ' between, the genes in the dataset and the canonical pathway is explained by chance alone.
  • P.AM calculates misclassificatio error rate in the training set of data for each of the two classes according to varying threshold (a unique statistical parameter), A threshold is chosen when the misclassifreation error is minimized for both classes to define a subset of probes from the entire training data set, designated as classifier probes. " These classifier probes were used for class prediction on testing data sets. Hierarchical clustering was used with the complete linkage- lgorithm to evaluate the accuracy of classification.
  • Mieroarray data was analyzed from lllumina Human HT12 Bead-Chips comprising -47,300 probes hybridized, with UNA samples extracted from whole blood specimens from 22 febrile children (8 positi e for huma herpesvirus 6 [MHV-6], 8 positive for adenovirus and 6 positive for enterovirus, and 8 with acute bacterial infection).
  • the same mieroarray assay was performed, on blood samples from 35 afebrile children (2 positive for HHV-6, 3 positive for adenovirus, 8 positive for riiinovirus, and 22 virus-negative control children).
  • probes sets were intersected that were significantly up- or down-regulated for each of the virus-positive groups and the febrile acute bacterial infection group compared to afebrile virus-negative, control children (FIG. 1A).
  • FIG. 1 illustrates identification of virus- and bacterial response-specific probes for distinguishing virus-positive febrile children and febrile children with acute bacterial infection from virus-negative afebrile children.
  • FIG. 1 A illustrates a Ven diagram showing identification of virus- and bacterial response-specific probes. Sets of probes differentially expressed in febrile children positi ve for 1 or more of the 3 viruses compared to vims- negative afebrile control children were intersected and 1.6 1 "panvirus" probes were identified and intersected with the set of probes thai were differentially expressed in children with febrile acute bacterial infection compared to virus-negative afebrile control children.
  • FIG. IB and FIG. I C illustrate heat-maps showing gene expression based on 260 viral response-specific probes ( FIG. 1 B) and 1321 bacterial response-specific probes (FIG. IC) in children with febrile and afebrile viral and bacterial infections and afebrile virus-negative control children. These probes were selected in (he same manner as the 413 vims -specific and 1939 bacterial response-specific probes described above except that for this selection, with the exclusion of 2 of 22 virus- negative controls. Probes that were not annotated in GenBank Build36 (National Center for Biotechnology Information) were also excluded. F+. febrile, afebrile. Each row represents a gene whose expression value is normalized to the mean of the afebrile virus-negative control group and each column represents one individual. Red in original represents up- regulation and blue represents down-regulation.
  • 260 probes with significant up- or down-regulation specifically In virus-positive febrile children and 1321 probes with significant up- or down-regulation specifically in children with febrile acute bacterial infection were identified.
  • Analysis of the 260 viral probes revealed overlap in gene expression profiles for febrile children who were positive for adenovirus, HHV-6, or enterovirus infection, which were very different from the profiles of most afebrile children (FIG. .1 B). Profiles of virus-positive and virus-negative afebrile children were indistinguishable.
  • Analysis using the 1321 bacteria! probes displayed similar patterns of gene expression for most of the children with, fever and acute bacterial infection that differed from those of the other groups, with a few exceptions (FIG. IC).
  • FIG. 2 illustrates blood leukocyte transcriptional profiles of febrile and afebrile HHV- 6-positive children compared to those of afebrile virus -negative children.
  • FIG. 2A illustrates clustering of probe sets with a statistically significant > 2-fold difference between HHV ⁇ 6-positive febrile children and afebrile virus-negative control children (FDR 5%).
  • FIG. 2B illustrates a principal component analysis of differentially expressed genes, with each oval representing one child.
  • FIGS. 2C-2F illustrates clustering of differentially expressed genes in FIG. 2A according to expression intensity in 4 Ingenuity® canonical pathways.
  • Each row represents a gene whose expression value is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column represents one individual. Red represents up-regulaiion and blue represents down- re g ulation.
  • FIG, 5 illustrates blood transcriptional profiles of febrile adenovirus-positive children as different from the profiles of healthy children and afebrile children with adeivoviros infections.
  • FIG. 5A illustrates clustering of probe sets with a statistically significant and greater than two-fold difference between adenovirus- positive febrile children and virus-negative afebrile controls (false discovery rate (FDR) at 5%).
  • FIG. 5 B illustrates a principal component analysis of differentially expressed genes, with each oval representing one child.
  • FIGS, 5C-5G illustrate clustering of differentially expressed genes from FIG. 5A according to expression intensity in five Ingenuity® canonical pathways.
  • Each row represents a gene with expressio value that is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column represents one individual. Red represents up-regtsSation, and blue represents down-regulation.
  • FIG, 6 illustrates blood transcriptional profiles of enterovirus-positive febrile children, as different from the profiles of virus-negative afebrile children.
  • FIG. 6A illustrates ciustering of probe sets with, a statistically significant greater than twofold difference between enterovirus-positive febrile children and virus-negative afebrile controls (P ⁇ 0.05, FDR at 20%).
  • FIG, 6B illustrates a principal component analysis of differentially expressed genes, with each oval representing one child.
  • FIGS. 6C-6G illustrates clustering of differentially expressed genes in FIG. 6A according to expression intensi ty in five Ingenuity® canonical pathways.
  • FIG. 7 illustrates blood transcriptional profiles of febrile children with acute bacterial infections as different from the profiles of vims-negative afebrile children.
  • FIG. 7 A illustrates clustering of probe sets with a statistically significant greater than two-fold difference between febrile children with acute bacterial infection and virus-negative afebrile controls (FDR. at 5%).
  • FIG. 7B illustrates a principal component analysis of differentially expressed genes, with each oval representing one child.
  • FIGS. 7C-7G illustrate clustering of differentially expressed genes from FIG. 7 A. according to expression intensity in five Ingenuity® canonical pathways. Each row represents a gene with an expression value that is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column represents one individual Red represents np-regulation, and blue represents down-regulai on.
  • FIG. 8 A and FIG. 8B Pathways with the most significant transcriptional changes for children with each of the three viral infections and with acute bacterial infection are shown hi FIG. 8 A and FIG. 8B.
  • FIG. 8A aftd FIG. 8B illustrate selected significantly up- and down-regulated
  • the pathways were arranged in ascending order by average P value of four infections for a pathwa (i.e., the most significantly up- or down-regulated pathway is at the top).
  • FIG. 2A A principal component analysis of the transcriptional profiles confirmed clear differences between the febrile and afebrile HHV-6-positive children (FIG. 2B).
  • FIG. 5 The gene expression profile of adenoviras-positive febrile children is illustrated in FIG. 5, Statistical comparison of transcriptional profiles between adeno virus-positive febrile children and virus-negative afebrile children showed 5604 probes with significant transcriptional changes including 847 with a 2-fold or greater Increase (576) or decrease (271 ) in expression level Principal component analysis confirmed the differences between the febrile and afebrile adenovirus-posi ive children, IFI27/ISG 12A was up-regulated. FIG. 8 displays the pathways with the significant transcriptional changes.
  • Transcriptional pathways were differentially activated in febrile children with viral and bacterial infections. Some Ingenuity® canonical pathways had significant transcriptional changes in febrile children positive for one of the three viruses (HHV-6, adenovirus, enterovirus) or with acute bacterial infection. Pathways thai were activated in each of the four infection groups were Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses, TREMl Signaling, and Toll-like Receptor Signaling. Some genes .trom the Natural Killer Cell Signaling pathway were down-regulated in each of the four infection groups. The Interferon Signaling Pathway and the Activation of interferon Regulatory Factors by
  • Cytosolie Pattern Recognition Receptors Pathway were more activated in febrile virus- positive children compared to febrile children with acute bacterial infection. In contrast, genes in the hitegrin Signaling Pathway were activated in bacterial infection. Transcriptional changes in each of these pathways are displayed in FIG. 8,
  • Table 2 These probes were had significant up- or down -regulation in children • positive for only one virus or with acute bacterial infection using an adjusted P value of 0.05. Gene transcription in children positive for only one virus and children with acute bacterial infection were each compared with gene transcription in afebrile virus-negative control, children.
  • Candidate genes were derived using the shrunken centroid algorithm procedure in the Prediction of Mi.eroar.ray Analysis tool from Stanford Universit (littp; /www- stat.stanibrd.edu/--tibs SAM ). Probes were sorted within each virus/bacteria grou by descending fold change (when up-regulated) or ascending fold change (when down- regulated). Bold and italic fonts indicate genes that at adjusted F value ⁇ 0.05.
  • FIG. 3 illustrates probes specific for individual viruses and for bacteria, in these investigations, viral -spec fic and bacterial response-specific probes were subjected to the shrunken centroid algorithm individually for each of the 4 pathogen groups to find a non- limiting reduced number of probes with ability t differentiate among pathogen groups.
  • Each row represents a probe and each, column displays probes for one febrile child positive for the indicated vims or with acute bacterial infection.
  • Classifier probes were identified to distinguish viral and bacterial infections in febrile children with validation on independent daiasets. Host transcriptional profiles unique to either viral or bacterial, infection were characterized to assist in making this clinical discrimination, individual-gene-based and pathway-based approaches for selecting probes were compared. For the gene-based approach * a "master set" was used, including the 1581. (260 viral- and 3321 bacterial-specific) probes described above and a limited subset of 38 of the 1581 selected using the shrunken centroid -algorithm.
  • the shrunken centroid algorithm was used to select 22 probes from the Interferon Signaling Pathway (selectively activated in virus-positive febrile children) and the Integrm Signaling Pathway (selectively activated in febrile children with acute bacterial infection).
  • the hybrid approach used 33 probes selected using the shrunken centroid algorithm from the master set and from the Interferon Signaling and Integrin Signaling Pathways, 9 classifiers were selected from the 3 sets of classifiers described above and validated by RT-qPCR. High correlation in expression level was found for 9 classifier genes between RT-qPCR and microarray results (FIG. 9, Table 3).
  • FIG. 4 illustrates classifier probes that discriminate febrile children positive for viruses from febrile children with acute bacterial infections.
  • FIG. 4A illustrates 1581 gene- based classifiers.
  • FIG. 4B illustrates 1.8 gene-based classifiers.
  • FIG. 4C illustrates 22 pathway-based classifiers.
  • FIG. 41 illustrates 33 classifiers selected from gene-based and pathway-based classifier sets. In each panel, patients are displayed as. columns and probes as rows. Gene symbols are shown in blue for bacterial infection-specific genes and in green for viral, infection-specific genes. Expression values presented in the heat map were normalized to the mean, of the afebrile virus-negative control cases.
  • Hierarchical clustering was used to classify patients into two groups with the majority of cases classified as either viral (green tree branch) or bacterial (blue tree branch). Classification as predicted using the K-nearest neighbor algorithm is shown as a bar above each heat map, with green showing classification as viral and blue showing classification as bacterial. True class was determined by virus- specific PG and bacterial cultures, and is designated by green (viral) or blue (bacterial) letters: A, adenovirus; B, Bacteria; E, enterovirus; II, IJHV-6. Classification based on patients' white blood cell (WBC) count is shown beneath each heat map. The upper strip shows classification based on age-specific norma! values and the lower strip shows
  • WBC white blood cell
  • the signal Intensity of the probes is shown in FIG. 9, and classification performance of each set is summarized in Table 4, Correct classificatio based on hierarchical clustering ranged from 77-90% and 83-90% based on die ⁇ nearest neighbor method.
  • FIG. 9 illustrates quanlile-no.rmalized raw signal intensity of classifier probes in 30 febrile children (22 virus-positive children and S children with acute bacterial, infection) in our study.
  • FIG. 9 A illustrates 18 -classifiers identified from 260 viral- arid 1,321 bacterial response-specific probes.
  • FIG. B illustrates 22 classifiers identified from 34 genes in the ingenuity® IFN signaling pathway and 205 genes in th Jngenuity® (Ingenuity Systems, inc., Redwood City, CA) integral signaling pathway
  • FIG- 9C illustrates 33 classifiers identified • from using both gene-level and pathway-based approaches.
  • FIG. 9D illustrates relative expression da ta of nine classifier genes, generated in quantita tive RT-PCR (RT-qPCR).
  • This example illustrates RT-qPCR validation assays.
  • RT-qPGR validation assays primers and probes of assay-on-demand were purchased from life Technologies (Applied Biosystems, Forster City, CA), and master mix was from Quanta Biosciences (Gaithersburg, MD) tor .reverse transcription and quantitative PCR (RT-qPCR) assays.
  • the assays were carried out in triplicate on an ABS 7500 real-time PGR. instrument following manufacturer's protocols. Assays had >80% PCR efficiency and ⁇ 15% coefficient of variance in triplicate reactions.
  • FIG. 1.0 illustrates validation of three sets of classifier probes discriminating virus- positive febrile children from febrile children, with acute bacterial infection using three independent cohorts of patients.
  • the 1 -sample validation set included. 18 subjects with influenza A, 29 subjects with Escherichia eoU, 31 subjects with Staphylococcus aureus, and J 3 subjects with Streptococcus pneumoniae. This set was profiled with the AiTyrneirix ( Santa Clara, California USA) Human Genome UI33A Array platform.
  • the 22-sample validation set included seven children with influenza A, three children with influenza B, six children with 5. aureus, and six children with & pneumonia.
  • This validation set was profiled with the Afiy etrix Human Genome U i 33 Plus 2.0 Array platform.
  • the 24-sample validation set included 5 subjects with influenza A, 3 subjects with influenza B, 13 subjects with '. aureus, and 3 subjects with S. pneumoniae, and it was profiled with the illoraina Sentrix Human-6 Expression BeadChip platform. Overall prediction, accuracy was 95% (130/137),
  • FIG. 1 OA a full set of 785 probes overlapped across all three datasets with 1 ,581 viral- and bacterial response-specific probes, FIG. I DS gene-based classifiers (n « 1 8), FIG. IOC pathway-based classifiers (n « 22), and FIG, !OD hybrid gene- and pathway-based classifiers (n ⁇ 33), respectively.
  • Patient groups are indicated by colored stripes at the top of the heat. map. True class indicates status determined by virus-specific PGR assays and bacterial cultures, and it was assigned to these cases in Ramilo, O..
  • Predicted class was determined by prediction made with the classifier probes, and it is labeled with green for viral or blue for bacterial infection.
  • Gene names in green signify genes selected from the viral -specific gene set (or the IFN signaling pathway genes), and gene names in blue represent genes selected from the bacterial-specific gene set (or the integral signaling pathway genes).
  • Expression values presented in the heat-maps were normalized to the mean of cases with bacterial infection within each datasef. Heat-map rows are gene probes, whereas columns are
  • This example illustrates utilizing the Pearson test to determine correlation between gene expression profiles and white blood cell counts / differentials.
  • FIG, 1 1 illustrates correlation of transcriptional changes and leukocyte subpopulations in febrile young children.
  • whole-blood samples were collected from 30 febrile children with .confirmed viral/bacterial infection.
  • Probe sets with at least a 1.5-fold change in level of expression over virus-negative afebrile controls are shown.
  • the expression pattern of the corresponding 4,716 probe sets is displayed in hierarchical cluster format, where rows represent genes and columns represent individual samples.
  • Correlation coefficients were calculated between the expression level of each probe set and white blood ceil, counts (total, neutrophil, lymphocyte, bands, and monocyte counts) across 30 patients.
  • the correlation values are plotted as moving averages of 50 probe sets (along the vertical axis). Dashed lines indicate the lowest values of correlation coefficients significant (adjusted P ⁇ 0.05) for each parameter.
  • This example illustrates diagnosis of a. symptomatic adenovirus infec tion in a subject.
  • a patient exhibits symptoms that initially appear attributable to a viral or bacterial infection.
  • a health practitioner obtains a whole blood sample* a nasopharyngeal sample, and a blood sample in a TempusTM Blood RNA Tube (Applied Biosystems, Carlsbad, California) from the subject.
  • A. health practitioner orders a pathogen culture and a. serum antibody detection test. The health practitioner add tionally orders a test to determine expression levels of 260 viral response-specific endogenous genes and 1321 bacterial response-specific endogenous genes by a microarray hybridization assay.
  • the pathogen culture is negative and the serum antibody test is positive for an adenovirus.
  • the microarray hybridization assay reveals an increase in expression of 165 of the 260 viral, response-specific endogenous genes, and a decrease of 1200 of the 1321 bacterial response-specific endogenous genes.
  • the patient is diagnosed with a symptomatic adenovirus infection.
  • This example illustrates diagnosis of a symptomatic bacterial infection in a subject.
  • a patient exhibits symptoms that initially appear attributable to a viral or bacterial infection.
  • a health practitioner obtains a. whole blood sample, a nasopharyngeal sample, and a blood sample in a TempusTM Blood RNA Tube (Applied Biosystems, Carlsbad, California) from the subject.
  • a health practitioner orders a pathogen culture and a serum antigen detection test.
  • the health practitioner additionally orders a test to determine expression levels of 260 viral response-specific endogenous genes and 13 1 bacterial response-specific endogenous genes by a microarray hybridization assay.
  • the pathogen culture is positive forS. aureus and the se um antibody test is negative.
  • the microarray hybridization assay reveals a decrease in expression of 206 of the 260 viral response-specific endogenous genes and an increase in 1 145 of the 1321 bacterial response-specific
  • the patient is diagnosed with a symptomatic bacterial infection, specifically S. aureus infection.
  • This example illustrates a method of diagnosis of Fever Without an Apparent Source in a subject.
  • a patient exhibits symptoms of Fever Without an Apparent Source.
  • a health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in a TempusTM Blood NA Tube (Applied Biosystems. Carlsbad, California) from the subject.
  • a health, practitioner orders a pathogen culture and a serum antibody detection test The health practitioner additionally orders a. test to determine expression levels of the following endogenous genes; OSBPL8, VEIL, ACTR2, MAP2 , F13A1 , PROS! , ITGB3, YL- , ITGA2B, ITGB5, GNGI 1 , EP300, ZYX, ARAP3, AGER, ITGAX, S .RLI, IFNGRL
  • the pathogen culture is negative and the serum antibody test is positive for an enterovirus.
  • the ' microarray hybridization assay reveals an increase in expression of IF GR1, JFNGR2, MT2A,
  • This example illustrates a method of diagnosis of Fever Without an Apparent Source in. a subject
  • a patient exhibits symptoms of Fever Wi thout an Apparent Source
  • a health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a ' blood sample in a TempusTM Blood RNA Tube (Applied Biosysiems, Carlsbad, California) from the subject
  • a health practitioner orders a pathogen culture and a serum antibody detection test.
  • the health practitioner additionally orders a test to determine expression levels of the following endogenous aenes: BARE ERAS. A.CTR2, NCK2. PIK3CB, MAP2K4. ITGA2B, HGB3.
  • microarray hybridization assay reveals an increase in expression of BA ! , IPNGRE STAT2, IFB5, MX. I, OAS!
  • IFiTIand IFIT3 and a decrease in expression of RRAS, ACTR2, NCK2, PIK3CB, MA.P2K4, ITGA2B, 3TGB3, MYL , MYL9, ITGB5, GNGI I, ZYX, ITGAX and ITGAM.
  • the patient is preliminarily diagnosed with an unknown and symptomatic viral, infection.
  • This example illustrates a method of distinguishing a viral-caused infection from a bacterial-cause infection or a combination thereof.
  • a patient exhibits symptoms that initially appear attributable to a viral or bacterial infection
  • a health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in a TempusTM Blood RNA Tube (Applied Biosysiems, Carlsbad, California) from, the subject
  • a health practitioner orders a pathogen, culture and a serum pathogen antigen detection test.
  • the health practitioner additionally orders a test to determine expression levels of the following endogenous genes: MYB9, ARAP3, CD IC, T2A, SPATS2L, IRF7, ZBP1, OAS2, OAS] , ISO 15, IFI6, HERC5, OAS3, RSAD2, OAS.L.
  • IF1T3, OTOF and IFI27 by microarray hybridization assay.
  • the pathogen culture is negative and the serum antigen test is positive for adenovirus.
  • the raicroarray hybridization assay reveals an increase in expression of CD 1C, T2A, SPATS2L, IRF7, ZBP1, OAS2, OAS!, IS015, 1F16, HERC5, OAS3, RSAD2, OAST, IFFB, O TOF and 1F127 and a decrease n expression of MYH9 and ARAP3,
  • the patient is diagnosed with a symptomatic viral infection.
  • This example illustrates a method of distinguishing a viral-caused infection from a bacterial-cause infection or a combination thereof.
  • a patient exhibits symptoms that initially appear attributable to a viral or bacterial infection.
  • a health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in a Ter pusTM Blood UNA Tube (Applied Biosystenis, Carlsbad, California) from the subject.
  • a health practitioner orders a pathogen culture and a serum pathogen antigen detection test.
  • the health practitioner additionally orders a test to determine expression levels of the following endogenous genes: MYH9.
  • I SAD2, OA.SL, IFIT3, OTOF and 1F127 by niicroarray hybridization assay The pathogen culture is positive for E.coli and the serum antigen, test is negati ve.
  • the microarray hybridization assay reveals a decrease in expression of CD N1C, MT2A, SPATS2L, 1RF7, ZBPI , OAS2, OAST ISG15, IF16, HERC5, OA.S3, R.SAD2, DASL, 1FIT3, OTOF and IF127 and an increase in expression of ⁇ 9 and ARAP3.
  • the patient is diagnosed with a symptomatic bacterial infection.
  • This example illustrates a . method of diagnosis of a pathogen-associated disease.
  • a patient exhibits symptoms that initially appear attributable to a viral or bacterial infection.
  • a health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in a TenipusTM Blood RNA Tube (Applied Biosyste is, Carlsbad, California) from the subject.
  • a health practitioner orders a pathoge culture and a serum pathogen antigen detection test.
  • a health practitioner orders at least one test to determine expression levels of the following endogenous genes: IFI27, iSG! 5, OTOF, IFIT3, 1TGAM, and ITGAX by raicroarray hybridization assay.
  • the pathogen culture is negative and the serum antigen test is positive for adenovirus.
  • the raicroarray hybridizatio assay reveals a decrease in expression ofJTGAM, and ITGAX and an increase in expression of IF.I27, ISGI S, OTOF, and IF1T3.
  • the patient is diagnosed with a symptomatic adenoviru infection.
  • This example illustrates a method of diagnosis of a pathogen-associated disease.
  • a patient exhibits symptoms that initially appear attributable to a viral or bacterial infection.
  • a health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in. a TemposTM Blood RNA Tube (Applied Biosystems, Carlsbad, California) from the subject.
  • a health practitioner orders a pathogen culture and a serum, pathogen antigen detection lest.
  • a health practitioner orders ai least one test to determine expression levels of the following endogenous genes: 1FI27, ISG15, OTOF, 1F1T3, ITGAM, and ITGAX by mic.roar.ray hybridization assay.
  • the pathoge culture is positive for B.coli and the serum antigen test is negative-
  • the microarray hybridization assay reveals an increase m expression of ITGAM, and ITGAX and a decrease in expression of IFI27, ISGl 5, OTOF, and ⁇ 3, The patient is diagnosed with a symptomatic bacterial infection.
  • This example illustrates a method of diagnosis of a viral pathogen-associated disease.
  • A. health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in a TempissTM Blood RNA Tube (Applied Biosystems, Carlsbad, California) from the subject.
  • a health practitioner orders a pathogen culture and a serum pathogen antigen detection test.
  • a health practitioner orders at least one test to detemiiiie expression levels of the following endogenous genes: IFI27, ISG.15, OTOF, IF1T3, ITGAM, and ITGAX by .microarray hybridization assay.
  • the pathogen culture is negative and the serum antigen test is positive for HHV-6.
  • the microarray hybridization assay reveals a. decrease in expression of ITGAM, and ITGAX and an increase in expression of IFI27, ISGl 5, OTOF, and IF1T3.
  • the patient is diagnosed with a symptomatic HHV-6 infection.
  • This example illustrates a method of diagnosis of a viral pathogen-associated disease
  • A. patient exhibits symptoms that initially appear attributable to a viral or bacterial infection.
  • a health practitioner obtains a. whole blood sample, a nasopharyngeal sample, and a blood sample in a TenipusTM Blood RNA Tube ( Applied Biosystems, Carlsbad, California) from the subject.
  • a health practitioner orders a pathogen culture, a serum pathogen antigen test, and serum antibody detection test.
  • the health praciitioner additionally orders a test to determine expression levels of the following endogenous genes: ⁇ 9, ARAP3, CDK IC, T2A, SPATS2L 1RF7, ZB.P1 , OAS2, OAS !
  • iS IS IF16, H.ER.C5, OAS3, RSAD2, OAST, IF1T3, OTOF and IF127 by microarray hybridization assay.
  • the pathogen culture is negative, the serum antibody test is negative, and the serum antigen test is positive for enterovirus.
  • the roi.croa.rray hybridization assay reveals an increase in expression of
  • CD NIC MT2A SPATS2L, IRF7, ZBF 1 ? OAS2, OASl , ISG15, W16, HB.RC5, AS3. RSAD2, OASL, ! ⁇ 3, OTOF and IFI27 and a decrease in expression of MYH9 and ARAP3.
  • the patient is diagnosed with a symptomatic enterovirus infection.
  • a patient exhibits symptoms that initially appear attributable to a viral or bacterial infection.
  • a health practitioner obtains a blood sample in a TempusTM Blood RNA. Tube (Applied Biosysteras, Carlsbad, California) from the subject The health practitioner orders a test to determine expression levels of the following endogenous genes: ⁇ » ARAP3 S CDKN IC, T2A, SPATS2L, IRF7, Z.BPI. OAS2, OAS l , ISG15. W16, HERC5, OA S3, RSAD2, OASL, IFIT3, OTOF and FI27 by niicroarray hybridization assay.
  • the microarray hybridization assay reveals an increase in expression of CDKN IC, MT2A, SPATS2L, IRF7, ZBPT OAS2, OASl, ISG15, IFI6, H.ERC5, OAS3, RSAD2, OAS I,, IFIT3, OTOF and IFI27 and a decrease in expression of MYH9 and ARAP3.
  • the patient is diagnosed with a symptomatic viral infection.
  • This example illustrates a method of diagnosis of a pathogen-associated disease.
  • a patient exhibits symptoms that initially appear attributable to a viral or bacterial infection.
  • a health practitioner obtains a blood sample in a TempusTM Blood RNA " l ube (Applied Biosysteras, Carlsbad, California) from the subject. The health practitioner orders a test to determine expression levels of the following endogenous genes: MYH9, ARA.P3, CDKNIC, MT2A, SPATS2L, IRF7, ZBP1, OAS2, OASl , ISO 15, IFI6, HER.C5, OAS 3, RSAD2, OASL, IF1T3, OTOF and 1FI27 by niicroarray hybridization assay.
  • the mic.roa.rray hybridization assay reveals a decrease in expression of CDKNIC.
  • the patient is diagnosed with a symptomatic bacterial infection.

Abstract

Disclosed are methods of diagnosis of a pathogen-associated disease. These methods comprise: providing a biological sample from a human subject; determining presence, absence and/or quantity of a bacterial pathogen, a viral pathogen, or a combination thereof, by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DNA and/or RNA detection test, or a combination thereof; determining in the sample, expression levels of at least one endogenous gene in which aberrant expression levels are associated with infection with a pathogen, by a microarray hybridization assay, RNA-seq assay, polymerase chain reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern, Northern, or Western blot assay, an ELISA or a combination thereof, whereby the subject can be diagnosed with the disease if the subject comprises the pathogen and an aberrant level of expression of the at least one gene.

Description

Diagnostic Methods for infectious Disease Using Endogenous Gene Expression
CROSS REFERENCE TO RELATED APPLICATION
This application .claims priority -from U.S. Provisional Application Serial No.
61/881,508 filed September 24. 2013, which is incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
Thi invention was made with government support under NTH Grants
1 UAH2AI083266-01 from the 1AID, UL1 RR024992 from the NIH-National Center tor Research Resources, and. the Training Grant. T32MD049338 from the MOID. The government lias certain rights in the invention,
INTRODUCTION
Despite absence of bacterial, infection, many febrile children are treated with antibiotics. Virus and bacteria interact with different pattern recognition receptors in circulating blood leukocytes, triggering specific host transcriptional programs mediating immune response. Unique transcriptional signatures can be defined that discriminate viral from bacterial causes of certain infectious disorders. Viral infections can be a cause of Fever Without an Apparent Source (FWS) in young children. Host transcriptional profiles can be useful if a pathogen is not detected or if the pathogenic role of a detected agent is in question .
Circulating blood leukocytes can react, to pathogens by recognizing pathogen-specific molecular patterns through pattern recognition receptors leading to up or down-regulation of the expression of host genes associated with immune functions (Takeuchi, O., et al. 2010, Cell 140: 805-820 and Thompson, M. ., et l. 201 1 , Viruses 3: 920-940). Host
transcriptional programs can be.- differentially activated with different pathogens (Pasehos, K., et al. 2010, Trends Microbiol J 8(10):439- 47).
Other studie have attempted to use host transcriptional signatures to distinguish respiratory viral infections from respiratory bacteriai infections using "acute viral respiratory signatures" (Ramilo, O., et al. 2007, Blood .1 9: 2066-2077; Ramilo, O., et al 2009, Cell Host Microbe 6: 199-200; Zaas, A. ., et al. 2009, Cell Host Microbe 6; 207-217).
Most transcriptional analysis has additionally been applied in other previous studies. Popper, B ., et al 2009, J. Infect. Dis. 200; 657-666, used specific gene expression profiles to distinguish Kawasaki disease from adenovirus infections, Ardura. ML, et al 2009, PLoS One 4(5):e5446, characterized acute invasive Λ' aureus infections. Berry, M.P., ei al 2010, Nature 466: 973-977, distinguished active tuberculosis -from other infectious and inflammatory diseases. Pankla, R., et al. 2009» Genome Biol 10: R1.27 distinguished, septicemic melioidosis from other causes. Stojanov, S., et al. .201 1, Proc. Nat l Acad, Set USA 1.08: 7148-7153, identified potential biomarkers to aid in distinguishing PFAPA flares from, asymptomatic intervals, HPF (hereditary periodic fevers) flares, and healthy controls. Loughtnan, J.A., ei l. J. Infect Bis. 2009, 199: 294-301, demonstrated staphylococcal gene expression and regulation directly in human tissue. Wang, Z.Q., et ai. J. Med. Virol 2012, 84: 1.254-1266, identified a gene expression profile to evaluate the effects of HAdV-36 on gen e tran sc i rion .
PCT/US2007/075713 (WO 2008/024642 A2) of Banchereau, J.F., ei al., describes specific gene expression biomarkers for the differential diagnosis of >$ aureus, E. calif and influenza infections. PCT/US.2 10/046042 (WO 201 1/066008 A2) of Banchereau, J.F., ei al, describes blood transcriptional signatures of active versus laten t mycobaeterium tuberculosis infections.
Sleeker, S.E., ei l. "Predicting serious bacterial infection in young children with Fever Without Apparent Source," Acta Paediatr. 20 1 90: 1.226-32 used predictors from patient history, examination, and laboratory tests to determine bacterial causes of some subjects with FWS. The laboratory tests were white blood ceil count, serum C-reactive protein and the presence of >70 white blood cells in urinalysis. Their diagnostic methods. do not include an analysis of changes in host gene expression.
The current clinical standard tor diagnosis of Fever Without an Apparent. Source is a white blood cel.! couni test using a cutoff of 15.000/nirrr as recommended by the American Academy of Pediatrics in their guideline for the management of febrile childre 0-36 months of age (Baraff, L.J ., et al. 1993, Ann. Emerg. Med. 22: 1 198-1.210). Previous studies indicate that white blood cell count is a inadequate tool for .distinguishing between viral, and bacteria! infection, a distinction often used to determine whether or not to treat the patient with antibiotics (Rucunsky, S.L., et l 2009, Acad. Emerg. Med. 1.6: 585-590 and Herz, A.M., ei al. 2006, Pedmtr. Infect. Dis. J. 25: 293-300). 30% of white blood cell count test results for diagnosis of .Fever Without an Apparent Source are inaccurate. There is thus a need for a more accurate methods of diagnosis for Fever Without an Apparent Source.
US Application number 11/268373 (US20080020379 l) of Agan, B. ., et al provides a specific set of host gene expression markers, for identification of pathogenic infections with 47 genes as the minimal number of genes to classify febrile versus non-febrile patients, Analysis of host transcriptional profiles has been applied to the diagnosis of inflammatory and hematological diseases. For example in Aliantaz,
Figure imgf000004_0001
et al, 2007, J, Exp. Med. 204: 2131 -2144; Aaroe, I, et al 2010, Bre t Cancer Res !2:R7; Al.kadeh, A.A., et l 2000, Nature 403: 5(53-51 1 ; and ChaussabeL D., ei al. 201. 1 , "Blood Transcriptional
Fingerprints to Assess the mmune Status of Human Subjects," Immun logic Signatures of Rejection, ed. Manncol FM (Springer, New York), pp 105-125,
The application of molecular vital detection tests to clinical medicine can detect asymptomatic as well as symptomatic infection (Colvin, J.M., et al 2012, Pediatrics 130: e 1.455- 1462) and has thus created a need to determine the clinical significance of the
detection of viral nucleic acid in an individual patient,
'.In recent, studies, infection with speci fic viruses has been confirmed in children with FWS (Colvin, J.M., et al 2012. Pediatrics 130(6): e 1455- 1462; WySie, .M., etal PloS One. 2 12;7(6);e27735; and His, X., et al Proc. Nat 'l Acad Set. USA. 2013 1 10:12792- 12797),
While some other previous studies provide clinical information in certain groups of subjects and for some particular diseases and/or infections, they can be limited in application and accuracy . Thus, there is a need, for more accurate diagnostic methods for diseases such as Fever Without an Apparent Source (FWS). Additionally, there is a. need for diagnostic methods for more accurately discriminating subjects with viral from bacterial infections, SUMMARY
The present inventor has developed a prognostic model for diagnosis of a pathogen- associated or pathogen-based disease in a subject. The expression levels of endogenous prognostic genes can. provide clinically beneficial assays as well as biological insights. In various embodiments, testing expression le vels of endogenous prognostic genes can be supplemented with assays to determine presence, absence, and/or quantity of a pathogen.
The present inventor discloses methods of di agnosis of a pathogen-associated disease in a subject Additionally disclosed are methods of distinguishing a viral -caused infection from a bacterial-caused infection or a combination thereof. Additionall disclosed arc methods of diagnosis of a viral pathogen-associated disease and methods of diagnosis of Fever Without an Apparent Source (FWS),
In various embodiments, the present teachings include: host transcriptional profiles that can distinguish symptomatic from asymptomatic viral infection; virus-specific transcriptional profiles for DNA and RNA viruses that cause systemic infection; and viral and bacterial -specific transeripiional profiles that can distinguish between infections caused by different pathogens- In various embodiments, diagnostic methods of the present teachings include detecting transcriptional changes in multiple endogenous genes in multiple pathways in febrile children who are infected with D A viruses. RNA viruses, or bacteria.
Host transcriptional analysis can be a clinically relevant testing method, and additionally, can supplement pathogen-based nucleic acid amplification assays. Host transcriptional analysis can provide results associated with etiology. In some embodiments, host transcriptional analysis can provide results associated with etiology even when no pathogens are confirmed from the direct detection testing for microbial pathogens.
In some embodiments, host blood transcriptional signatures associated with categorie of infectious etiology, can be defined in a subject with FWS, such as a young child with FWS, In some embodiments, host blood transcriptional signatures can be of increased accuracy and predictive value than white blood ceil count-based criteria alone in
discriminating a febrile child with a viral infection from a febrile child with a bacterial infection. In some embodiments, host blood transcriptional signatures can be a supplement to tests that detect the presence of a possible pathogen but do not address its pathogenic role in the subject. In some embodiments, some genes of the Interfero Signaling Pathway can be uniquely activated in a febrile child with a viral infection, in some embodiments, some genes of the Integrin Signaling Pathway can be uniquely activated in a child with a bacterial infection. In some embodiments, host transcriptional profiles can be used to classify a febrile child with a viral or a bacterial infection with more accurac than a blood white blood cell (WBC) count.
In some embodiments, the present teachings include methods of diagnosis of a pathogen-based disease in a subject, and methods of determining etiology of a pathogen- based disease in a subject, in various configurations, these methods comprise: a) providing at least one biological sample from a human subject; b) determining presence, absence and/or quantity of a bacterial pathogen, a viral pathogen, or a combination thereof, by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DMA detection test, a pathogen RNA detection test, or a combination thereof; and c) determining in the at least one sample, expression levels of at least one endogenous gene in which aberrant expression levels are associated with infection with a pathogen, by a microarray hybridization assay, an NA-seq assay, a polymerase chain reaction assay, a LAMP assay, a Hgase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay, an enzyme- linked immunosorbent assay (ELI SA) or a combination thereof, whereby the subject is diagnosed with the disease if the subject comprises the pathogen and the sub ject has an aberrant level of expression of at least one gene associated with aberrant expression levels associated with infection with the paihogen. In some configurations, the at least one endogenous gene can consist of one gene,. In some configurations, the at least one endogenous gene can consist of two genes. In some configurations, the at least one endogenou gene can consist of three genes, in some configurations, the at least one endogenous gene can consist of four genes, in some configurations, the at least one endogenous gene can consist of five genes. In some configurations, the at least one endogenous gene can consist of six genes, in some configurations, the at least one endogenous gene can consist of seven genes, in some configurations, the at. least one endogenous gene can consist of eight genes. In some configurations, the. at least one endogenous gene can consist of nine genes. In some configurations, the at. least one endogenous gene can consist of ten genes. In some configurations, the at least one endogenous gene can consist, of eleven genes. In some configurations, the at least, one endogenous gene can consist of twelve genes. In some configurations, the at least one endogenous gene can consist of thirtee genes, i some configurations, the at least one endogenous gene can consist of fourteen genes, in some configurations, the at. least, one endogenous gene can consist of fifteen genes. In some configurations, the at least, one endogenous gene can. consist of sixteen genes. In some configurations, the at least one endogenous gene can consist of seventeen genes, in some configurations, the at least one endogenous gene can consist of eighteen genes. In some configurations, the at least one endogenous gene can consist of nineteen genes. In some configurations, the at least one endogenous gene can. consist of twen t genes, in some configurations, the at least one endogenou gene can consist of 21. genes. In some configurations, the at least one endogenous gene can consist of 22 genes. In some configurations, the at least one endogenous gene can consist of 23 genes. In some configurations, the at least one endogenous gene can consist of 24 genes. In some configurations, the at. least, one endogenou gene can consist of 25 genes. In some configurations, the at least one endogenous gene can consist of 26 genes. In some configurations, the at least one endogenous gene can consist of 27 genes. In some configurations, the at least one endogenous gene can consist of 28 genes. In some configurations, the at least one endogenous gene can consist of 29 genes. I some configurations, the at least one endogenous gene can consist of 30 genes. In some configurations, the at least one endogenous gene can consist of 31 genes. In some configurations, the at least one endogenous gene can consist of 32 genes, hi some configurations, the at least one endogenous gene can consist of 33 genes. In some configurations, the disease can be Fever Without an Apparent Source. In some configurations, the pathogen can be a virus. In some configurations, the virus can be selected from the group consisting of an adenovirus, an enterovirus, a human herpesvirus 6 (HBV-6) and a rhiiiovims. In some configurations, the pathogen can be a bacterium. In some configurations, the bacterium can be selected from the group consisting of an Escherichia coil, a Staphylococcus aureus a Streptococcus
pneumoniae and a combination thereof. In some configurations, the at least one endogenous gene can be selected from the group consisting of IFI27, ISO 15, OTOF, 1FIT3, ITGAM and 1TGAX. In some configurations, the at; least one gene can comprise, consist essentially of, or consist of OTOF and JTGAX. In some configurations, the at least one gene can comprise, consist essentially of, or consist of IFI27 and ITGAM. In some configurations, the
determining expression levels ca comprise, consist essentially of, or consist of a real time polymerase chain reaction assay, a reverse transcriptase polymerase chain reaction assay, or a combination thereof. In some configurations, the determining expression levels can comprise, consist essentially of, or consist of an oligonucleotide array assay, a probe hybridization assay, a gene expression array assay, a cDNA mieroarray hybridization assay or a
combination thereof, in some configurations, the microarray hybridization assay can comprise, consist essentiall of, or consist of from .1 to 47,300 probes, or about 47,300 probes, In some configuration , the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 5700 probes, or about 5700 probes. In some
configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 50 probes, or about 50 probes. In some con figurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 10 probes, or about 10 probes, in some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of between 18 and 33 probes, or about 33 probes, in some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from I to 260 probes, or about 260 probes. In some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 1321 probes, or about 1321 probes, in some configurations, the .microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 18 probes, or about I S probes. In some configurations, the microarray hybridization assay can comprise, consist essentially of or consist of from 1. to 22 probes, or about 22 probes. In some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 33 probes, or about 33 probes, in some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 260 viral response-specific probes or about 260 viral response-specific probes and from 1 to 1.321 bacterial response-specific probes or about I 321 bacteria! response-specific probes, in some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from to 3467 HHV-6 response- specific probes or about 3467 HHV-6 response-specific probes. In some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 464.HHV-6 response-specific probes or about 464 HHV-6 response-specific probes. In some configurations, the microarray hybridization assay can. comprise, consist essentially of, or consist of from 1 to 798 HHV-6 response-specific probes or about 798 HHV-6 response- specific probes. In some configurations, the microarray hybridization assay can comprise, consist essentiall of, or consist of from 1 to 5604 adenovirus response-specific probes or about 5604 adenovirus response-specific probes. In some configurations, the microarray h bridization assay can comprise, consist essentially of, or consist of from I to 2078 adenovirus response- specific probes or about 2078 adenovirus response-specific probes. In some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1 to 847 adenovirus response-specific probes or about 847 adenovirus response-specific probes, in some configurations, the microarray hybridization assay can comprise, consist essential ly of, or consist of from 1 to 4184 enterovirus response-specific probes or about 4184 enterovirus response-specific probes. In some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1. to 594 enterovirus response-specific probes or about 594 enterovirus response-specific probes. In some configurations, the microarray hybridization assay ca comprise, consist essentially of, or consist of from 1 to 678 enterovirus response-specific probes or about 678 enterovirus response-specific probes. In some configurations, the microarray hybridization assay can comprise, consist essentially of, or consist of from 1. to 1.234 bacterial response-specific probes or about 1234 bacterial response-specific probes. In some configurations, the microarray hybridization, assay can comprise, consist essentially of or consist of from I to 1939 bacterial response-specific probes or about. 1 39 bacterial response-specific probes. In some configurations, the at least one biological sample can be selected from the group consisting of peripheral blood mononuclear cells, a nasopharyngeal sample, a urine sample, a blood sample, a lumbar puncture sample, bodil fluid, a biopsy sample, a tissue sample and a combination thereof. In some configurations, the at least one biological sample can comprise, consist essentially of, or consist of a peripheral blood sample. In some
configurations, the at least one biological sample can comprise, consist essentially of, or consist of peripheral blood mononuclear cells, in some configurations, the at least one endogenous -gene can be selected from the group consisting of Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses Pathway genes, TREMI Signaling Pathway genes, Toll-like Receptor Signaling Pathway genes, Natural Killer Cell Signaling Pathway genes, Interferon Signaling Pathway genes, Activation, of Interferon Regulatory Factors by Cytosolic Pattern Recognition Receptors Pathway genes, Integra Signaling Pathway genes and a combination thereof, in. some configurations, the at least one endogenous gene can comprise, consist essentially of, or consist of an interferon Signaling Pathway gene. In some configurations, the at least one endogenous gene can comprise, consist essentially of, or consist of an tegrin Signaling Pathway geae. In some configurations, the human subject can be a human child, such as, without limitation, a human subject between 0 to 36 months of age. In some configurations, the human subject can be a human child, such as, without limitation, human subject between 2 to 36 months of age.
In some embodiments, the present teachings include methods of distinguishing a viral-caused infection from a bacterial-caused infection or a combination thereof in a subjec in various configurations, these methods can comprise: a) obtaining at least one biological sample from a human subject; b) determining presence, absence and/or quantity of a viral pathogen by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DNA detection test, a pathogen RNA detection test or a combination thereof; c) determining presence, absence and/or quantity of a bacterial pathogen by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DNA detection test, a pathogen RNA detection test or a combination thereof; and. d) determining in. the at least one sample, expression level of at least one endogenous gene hi which aberrant expression is associated with infection with a viral pathogen, and. expression level of at least one endogenous gene in which aberrant expression is associated infection with, a bacterial pathogen, by an assay selected from the group consisting of a microarrav hybridization assay, an A-seq assay, a polymerase chain reaction assay, a LAMP assay, a Hgase chain, reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay, an enzyme-linked immunosorbent assa and a combination thereof, whereby the subject is diagnosed with a viral infection if the sample comprises a) a virus pathogenic for the infection and b) aberrant expression level of at least one endogenous gene associa ted with a viral infection, and whereby the subject is diagnosed with a bacterial infection if the sample comprises a) a bacterium pathogenic for the Infection and b) aberrant expression level, of at least one endogenous gene associated with a acterial infection. In some configurations, the at least one gene associated with, a bacterial infection can be selected from the group consisting of FYN, CD247, EITPR3, CD3, ZAP70, PiXGl , PRKCFi, LCK, i T, PRKCQ Π Κ, RHOU, GNA13, PPPIR12A, RHOTl, FCER IG, LYN, RALB, GNAQ, MARC S, TGM2,
ARHGEFI 1, MYL1.2 , EP30Q, YL9. GREB5, FCGR2A, GNG1 , GNGl 1 , CI QB, OD2, TLR2, TLR I , RNASEL, C5AR1 , TLR.4, MYD88, PIK3CB, C3ARI , TLR6, CASPl , TLR5, LRC4, TLR8, IL i B, ITGB7, TSPAN4, PPP!Ri , ZYX, VASP, ITGA2B, ΪΤΟΒ5, VCL, ITGB3, MYLK, AS API , ITGAM, ΓΓΟΑΧ, KLRDI, KIR2DL3, .1R2DL4, KIR3DL3, K1R3DLL HCST, CD247, NCR3, FCGR3B, SIGLEC9, FCE IG, JAK2, CASP5 and a combination thereof, in some configurations, the at least one gene associated with a bacterial infection can be selected from the group consisting of RHOU, GNA13, PP 1R12A, .RHOTI. FCERI G, LYN, RALB, GNAQ, ARCKS, TGM2, ARHGEFI ί , ΜΥΙΛ 2A, EP300, MYL9, CREB5, FCGR2 . GNGl 0, GNGl 1 , CTQB, NOD2, TLR2, TLRI , RNASEL, C5 .RL TLR4, MYD88, PIK3CB, C3AR1 , TLR6, CASPl , TLR5, NLRC4, TLR8, ILI B, PPPIRl , ZYX, MYL 12A, VASP, ITGA2B, ITGB5, VCL, ITGB3, MYLK, ASAP I, STGAM, HOAX, FCGR3B, SIGLEC9, FCERIG, XAK2, CASP5 and a combination thereof, wherein the at least one gene has an aberrant level of expression of at least a 1.5 fold increase compared to a control level in some configurations, the at least one gene associated with a bacterial infection can be selected from, the group consisting of FYN , CD247, EFi'PRS, CD3, ZA 70, PLCGi , PRKCH, LCK, LAT, PRKCQ, ITK, ITGB7, TSPAN4, KLRDI, KiR2DL3,
K1R2DL4, KJR3DL3, KIR3DL! , HCST, CD247, NCR3 and a combination thereof, wherein the at least one gene has an aberrant level of expression of at least a 1.5 fold decrease compared to a. control level, m some configurations, the at least one gene associated with a bacterial infection, can be an Annexin A3 gene. In various configurations, if the disease is determined to be due to a bacterial infection, the patient can be treated by routine methods such as administratio of an antibiotic, which can be any antibiotic known to skilled artisans. In various configurations, if the disease is determined to be due to a viral-caused infection, the patient can be treated by routine methods such as administration of an anti-viral drug, which can. be any anti-viral drag known to skilled artisans,
In. some embodiments, the present teachings include methods of diagnosing or determining etiology of Fever Without an Apparent Source in a subject I» various configurations, these methods can comprise: a) providing at least one biological sample from a human subject; b) determining, presence, absence and/or quantity of a bacterial pathogen, viral pathogen, or a combination thereof, by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DNA detection test, a pathogen RNA detection test, o.r a combination thereof; c) determinin in the sample, expression levels of at least one endogenous gene associaied with aberrant expression levels resulting from inlection with the pathogen, by a microarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay, an enzyme- linked immunosorbent assay or a combination thereof, whereby the subject is diagnosed with the disease if the sample comprises the pathogen and the sample exhibits an aberrant level of expression of at least one gene associated with aberrant expression levels resulting from infection with the pathogen. In various configurations, if the FWS is determined to be due to a bacteria! infection, the patient can be treated, by routine methods such as administration of an antibiotic. In various configurations, if the disease is determined to be due to a viral infection, the patient can be treated by routine methods such as administration of an anti-viral drug, in various configurations, the sample ca be a. peripheral blood sample, a nasopharyngeal sample, a urine sample, a blood sample, a lumbar puncture sample, a bodily fluid, a biopsy sample, a tissue sample or a combination thereof
In some embodiments, the present teachings include methods of diagnosing or determining etiology of a pathogen- associated disease, in various configurations, these methods can comprise; a) providing at least one biological sample from a human subject; b) determining presence, absence and/or quantity of a bacterial pathogen, viral pathogen, or a combination thereof, by an assay selec ted from the group consisting of a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen D A detection test, a pathogen RNA detection test and a. combination thereof; e) determining in the sample, expression levels of at least one endogenous gene selected from the group consisting of IFI27, ISGiS, GTOF, O B, ITGAM, ITGAX and a combination thereof; by an assay selected from the group consisting of a microarray hybridizatio assay, an NA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay, an enzyme-linked immunosorbent assay, or a combination thereof, whereby the subject is diagnosed with the disease if the sample comprises tire pathogen and. the sample exhibits an aberrant level of expression of at least one gene selected from the group consisting of IF 127, ISO 15, OTOF, ίΡΓΠ, ITGAM, ITGAX and a combination thereof.
In some embodiments, the present teachings include methods of diagnosing a viral pathogen-associated disease. In various configurations, these methods can comprise: a.) providing at least one biological sample from a human subject; b) determining presence, absence and/or quantity of a virus selected from the group consisting of an adenovirus,, an enterovirus, HHV-6V or a combination thereof, by a pathogen culture, a serum, antibody detection test, a pathogen antigen detection test, a pathogen DMA detection test, a pathogen R A detection test, or a combination, thereof; c) determining in the sample, expression level of at least one endogenous gene that exhibits aberrant expression during infection with the virus, by an assay selected from the group consisting of a microarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain, reaction assay, a. Southern blot assay, a Northern blot assay, Western blot assay, an. enzyme-Unked immunosorbent assay and a combination thereof, whereby the subject, is diagnosed with the disease if the sample comprises a) the virus and b) an aberrant expression level of the at least one gene.
In some em bodiments, the present teachings include methods of diagnosing a
pathogen-associated disease, in various configurations, these methods can comprise: a) obtaining at least one biological sample from a human subject: and b) determining in the sample, expression levels of at least one endogenous gene selected from the group consisting of I I27, ISCi i 5, OTOF, M'B, ITGAM, ITGAX and a combination thereof; by a microarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay, an enzyme-linked immunosorbent assay or a combination thereof, whereby the subject is diagnosed with the disease if the sample exhibits an aberrant level of expression of at least one gene selected from the group consisting of IFI27, iSGl 5, OTOF, IFIT3, ITGAM,
ITGAX, and a combination thereof In some configurations, the disease can be Fever Withoui an Apparent Source. In some configurations, the biological sample can comprise peripheral blood mononuclear cells.
In various configurations of the embodiments, the at least one aberrant level of expression can be at least a 2 fold increase or decrease compared to a control level, in various configurations of the embodiments, the at least one aberrant level of expression can be the at least one aberrant level of expression is at least a 1 ,5 fold increase or decrease compared to a control level.
In various configurations of the embodiments, the at least one gene associated wi th an adenoviral infection can be selected from the group consisting of ATM, PRKCH, PR CQ, C1QB, C!QC, IRF7, OAS3, QAS1 , OAS2, AK2, EIF2, 1FIH 1, DDX58, NOD2, TLR5,
"NLRC4, TLR8, C3AR1 , IL1 B, TLR1 , TLR4, TLR6, MYD88, CASP!, IF1T3, 1FI35, BCL2, MED 14, IFNG.R2, IFNARI , IRF1, BAX, IRF9, PSMB8, IFITMI , JA 2, STAT2, TAP1 , IKBKB, IKBKAP, K1AAI271, TRAP6, TNF, TB l, TANK, IRF9, NFKBIA, 1RF7, 1SGIS, ADAR. ZB L il l 12; EOS, EY96, TLR5, IRA .3, TLR8, ΕΪΕ2Α 2, CD14, MAP J4, STAT4, HS.572649, CCR7, CD40LG, LIB, HLA-DOA, CREB5, FCGR3B, PCGR2A, IL1RN, LI 'BR, TYROBP, FCER.1G, FCGRiA, FCGR1 B and a combination thereof.
In various configurations of the embodiments, the at least one gene associated with a» adenoviral infection can be selected from the group consisting ofClQB, CIQC, 1RF7, OAS3, OA.S1, OAS2, AK2, EIF2, IF!H!, DDX58, NOD2, TLR5, ' LR.C4, TERS, C3AR1, IL!B, TLRL TLR4, TLR6, MYD88, CASPI, IFIT3, 1PF35, BCL2, IF ARI, IRFL BAX, 1RF , PSMB8, IFITM1, JAK2, STAT2, TAP.1, TRAF6, TNF, TBKl, 'FANE, IRF9,
NFKBIA, 1RF7, ISG15, ADAR, ZBP1, IFIT2, FOS, LY96, TLR5, .IRAK3, TLRS, EIF2A 2, CD 14, MAPK14, CREB5, FCGR3B, FCGR2A, IL1RN, LTBR, TYROBP, FCERIG, FCG IA, FCXI IB and a combination thereof, wherein the at least one gene has an aberrant level of expressio of at least a.1,5 fold increase compared to a control level
In various configurations of the embodiments, the at least one gene associated with an adenoviral infection can be selected from the group consisting of ATM, PRKCFL PRKLCQ, MED 14, IFNGR2, IK.BKB, IKBKAP, K1AA127I, STAT4, HS.572649, CCR7, CD40LG, LTB, HLA-DO.A and a combination thereof, wherein the at least one gene has an aberrant level of expression of at least a 1.5 ibid decrease compared to a control level.
In various configurations of the embodiments, the at least one gene associated with an enterovirai infection can be selected from, the group consisting of NLRC4, TLR8, TLR5, NOD2, KNASEL, TLR2, 1'LRl, C5A 1, MYD88, TLR6, CASPI, IL1B, CiQB, 1RF7, OASL OAS3, EIF2A 2, DDXS8, IFtFit, OAS2, !FITl, ΠΠ3, 1F135, MXL TAP I, STAT2, IFITM L STALL JAK2, IRF7, ISG1.5, 1FIT2, DHX58, ZBPL FOS, LY96, MAP2K3, MAPK14, CASP5 and a combination thereof
hi various configurations of the embodiments, the at least one gene associated with an enterovirai infection can be selected from the group consisting of NLRC4, TLR8, TLR5, NOD2, RNASEL, TLR2, TJLRl, C5AR.1, MYD88, TLR.6, CASPI, IE IB, CIQB, IRF7, OASI, OAS3, B1F2A , DDX58, IFIHL OAS2, IFITL IFFF3, IFI35, M l, TAP 1, STAT2, IFITMJ, STATi, JAK2, IRF7, SSGI5, IF.IT2, DHX5S, ZBPL FOS, EY96, M P2K3, MAF I4, CASP5 and a combination thereof, wherein the at least one gene has an aberrant level of expression of at least a.1,5 fold increase compared to a control level,
In various configurations of the embodiments, the at least one gene associated with an BHV-6 infection can be selected from the group consisting of CCL5, PRKCH, TLR5, TLR2, NOD2, TLR8, TLR4, TLR6, C QA, MYD88, R1PK2, !LIB, C3AR1, CASP1, CTQB, CIQC, OASURF7, OAS3, EIF2A 2, TLR7, OAS2, DDX58, IFffl L IFIT3, IFITI, SOCSL MX1 , IFI35, IFITM1 , TAP! , STAT2, JAK2, SI' ATI, TY OBP, CD86, IL1B, CASP5, CCL2, TLR9, TR F6, IRAK 4, CHtJK, CD 14, MYDS8, TLR I, FOS, LY96 and a combination thereof.
In various configurations of the embodiments, the at least one gene associated with an BHV-6 infection can be selected from the group consisting of TLR5, TLR2, MOD2, TXR8, TLR.4, TLR.6, C.1.QA, YDS8, R.1PK2, [LIB, C3AR.1 , CASP1 , C1QB, CIQC, OAS1, 1RF7, OAS3, EIF2AK2, TLR7, OAS2, DDX58, IF!HL IFIT3, IFITI, SOCS l, MX1 JFI3S, IFSTMl, TAPL STAT2, JAK2, STAT.1 , TYR.OBP. CD86, ΪΠ Β, CASP5, CCL2, TRAF6, IRA 4, CHUK, CD 14, MYD88, TLRL FOS, LY96 and a combination thereof, wherein the at least one gene has an aberrant level of expression of a! least a 1 ,5 ibid increase compared to a control level.
In various configurations of the embodiments, the at least one gene associated with an HHV-6 infection can be selected from the group consisting of CCL5, FRKCH and TLR9, wherein the at least one gene has an aberrant level of expression of ai least a 1.5 fold decrease compared to a control level. In some configurations, the at least one gene can he selected from the group consisting of MYFI9, ARAP3, CD Ni C, MT2 , SPATS2L, IRF7, ZBPl , OAS2, OASL ISG15, IF16, HERC5, OAS3, RSAD2, OASL, IFIT3, OTOF and IFI27.
In various configurations of the embodiments, the at least one gene associated with a viral infection can be selected from the group consisting of CDK.N 1 C. MT2A, SPATS2L, IRF7, ZBPl , OAS2, OASI , LSG15, IF16, HERC5, OAS3, RSAD2, OASL, IP II 3, OTOF and IFI 27, wherein the at. least one gene has an aberrant level of expression of at l east a 1.5 fold increase compared to a bacterium-infected control level. In some configurations, the at least one gene can be selected from the group consisting of MYH9 and ARA.P3, wherein the ai least one gene has an aberrant level of expression of at least a 1.5 fold decrease compared to a bacterium-infected control level In some configurations, the at least one gene can be selected from the group consisting of BAK1 , R AS, ACTR2, NCK2, PIK3CB, MAP2K4, ITGA2B, ITGB3, MYLK, YL9, 1TGB5, GNG I 1 , ZYX, STGAX, IFNGRl , ITGAM, STAT2, IFI35, MX J , OASI , IFITI and SF1T3,
In various configurations of the embodiments, the at least one gene associated wi th a viral infectio can be selected from the group consisting of 8AKL IFNGRl, STAT2, IF135, MX I, OAS I , IFITI and - IFIT3, wherein the at least one gene has an aberrant level of
expression of at least a 1.5 fold increase compared to a bacterium-infected control level, in some configurations, the at least one gene can be selected from the group consisting of RRAS, ACTR2, NCK2, PIK3CB, AP2K4, 1TGA2B, 1TGB3, MYLf MYL9, ITGB5, GNG 11. ZYX. ITGAX and JTGAM, wherein the at least one gene has an aberrant level of expression, of at least a 1.5 fold decrease compared to a bacterium -infected control level In some configurations, the at least one gene can be selected from, the group consisting of OSBPL8, VHL, ACTR2, MAP2 4, FBA 1, PRO i, ITOB3, MYL9, .1TGA2B, ITGB5, GNG 1 1 , EP300, ZYX, ARAP3, AGER, ITGAX, SORL1, IFNG l, IFNGR2, ITGAM, MT2A, SPATS2L, OAS2, OAS.l , 1SG15, IFI6, ΕΤΠ , HERC5, OAS3, RSAD2, OASL OTOF and IFI27. In some configurations, the at least one gene can be selected from the group consisting of lFNGRI , IFNGR2, MT2A, SPA.TS2L, OAS2, GAS1, ISG15, IFI6, 1FIT1 , HERC5, OAS3, RSAD2, OASL, OTOF and IFI27, wherein the at least one gene has an aberrant level of expression of at least a. 1.5 fold increase compared to a bacterium- infected control level. In some configurations, the at least one gene can be selected fro.ra the group consisting of OSBPL8, VHL, ACTR2, MAP2K4, FI3AI, PROS! , ITGB3, MYL9, ITGA2B, iT'GBS, GNG! lf EP300, ZYX, A AP3, AGER, ITGAX, SORU and IGTAM, wherein the at least one gene has an aberrant level of expression of ai least a 1.5 fold decrease compared to a bacterium-infected control level. In some configurations, the at least one gene can be selected from the group consisting of iFI'27, iSG! S, OTOF and 1FIT3, whereby the subject s diagnosed with a viral infection if the subject 'has an aberrant level of expression of at least a 1 ,5 fold increase of at least on gene selected from ihe grou consisting of ΙΡΪ27, 1SG15, OTOF and IFIT3, compared to bacterial-infected control level, in some
configurations, the at least one gene can be selected from the group consisting of 1FI27, 1FIT3, and a combination thereof, whereb the subject can be diagnosed with febrile HHV-6 if the subject has an aberrant level of expression of at least a 1.5 fold increase of the at least one gene selected from the group consisting of IFI27 and. IFFF3, compared, to an afebrile HHV-6 control level or a febrile control level In sotne configurations, ihe ai least one gene can be selected from the group consisting of IPI27, ISG15, and a combination thereof, whereby the subject ca be diagnosed with, febrile adenovirus if the subject has an. aberrant level of expression of at least a 1.5 fold increase of the at least one gene selected from the group consisting of IFI27 and ISO 15, compared to an afebrile adenovirus control level or a febrile control level in some configurations, the at least one gene can be selected from the group consisting of IF 127, ISO 15 and 1FIT3, whereby the subject is diagnosed with febri le enterovirus if the subject has an aberrant level of expression of at least a 1 ,5 fold increase of the at. least one gene selected from the group consisting of IFI 27, ISO 15 and IFIT3, compared to an afebrile enterovirus control level or a febrile control level, hi some configurations, the at least one gene can be selected from the group consisting of ITGAM and ITGAX, whereby the subject can be diagnosed, with a bacteria.1 infection if the subject has an aberrant level of expression of at least a 1.5 fold increase of the at least one gene selected from the group consisting of ΓΓΟΑΜ and TIG AX, compared to an afebrile control level or a viral-infected control level
In various configurations of the embodiments, diagnostic acc uracy can be at least 70%. M various configurations of the embodiments, diagnostic accuracy can be at least 75%. in various configurations of the embodiments, diagnostic accuracy can be at. least. 80%, In various configurations of the embodiments, diagnostic accuracy can be at least 85%. In various configurations of the embodiments, diagnostic accuracy can be at least 90%.
'.In some embodiments, the present teachings include methods of determining etiology of a disease, such as, without limitation., Fever Without an Apparent Source (FWS) in a subject such as. for example, a human child. In various configurations, these methods can include a) providing a peripheral blood sample from a human subject; b) determining presence, absence and/or quantity of a bacterial pathogen, a viral pathogen, or a combination thereof, by a pathoge culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DNA detection test, a pathogen RN A detection test, or a combination thereof; c) determining in the sample, expression levels of at least one endogenous gene in which aberrant expression levels are associated with infection with a pathogen, by a mieroarray hybridization assay, an RN A-seq assay, a polymerase chain, reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay, an enzyme-linked immunosorbent assay (ELISA) or a combination thereof, wherein the etiology of the disease I determined if the subject comprises the pathogen and the subject has an aberrant level of expression of at least 1.5 fold, increase or decrease of at least one gene, wherein an aberrant level of expression of the at least one gene is associated with infection with the pathogen.
In various aspects, the present teachings include therapies selected on the basis of a transcriptional profile determined by the disclosed methods. These can include, without limitation, selection and administratio of an appropriate antibiotic in the. case of a disease determined to he caused by a bacterial infection, or selection, and administration of an appropriate anti-viral drug in the case of a disease determined to be caused by a viral infection.
'In some embodiments, the present teachings include an antibiotic for use in a method of treatment of Fever Without an Apparent Source (FWS) in a subject, characterized In that the subject has in a biological sample such as a peripheral blood sample a least a .1 ,5 fold increase in expression level compared to a control level of at least one endogenous gene, such as, without limitation, at leas one endogenous gene selected from the grou consisting FYR, CD247, EITPR3, CD3, ZAP7Q, PLCG1 , PRKCH, LC , LA I; PRKCQ, ΓΓΚ, RHOU, G A13, PPPl R12A. RHOTI, FCER.lG, LYN, RALB, GNAQ, MARC S, TGM2,
ARHGEFH, MYLI 2 , EP300, MYL9, CRBB5, FCGR2A, GNG10, GNGl 1 , CI QB, NOD2, T.LR2, TLR.i , RNASEL, C5AR1, TLR4 MYD8S, PIK3CB, C3AR1, TLR6, CASPi, TLR5, NLRC4, TLR8, ΪΠΒ, ITGB7, TSPAN4, PP I 1, ZYX, VASP, ITGA2B, ITG.B5, YCL, 1TGB3, MYIK, ASAP I , FfGAM, ITGAX, I.RD1 , KIR2DL3, KIR2DL4, KJR3DL3,
KIR3DL1, HCST, CD247, NCR3. FCGR3B, S1GLEC9, FCERIG, JA 2, CASP5 and a combination thereof.
In some, embodiments, the present teachings include an antibiotic for use in a method of treatment of a disease caused by bacteria, characterized in that the patient has at least a 1 ,5 fold increase in transcription levels of one or more genes that increase in expression level upon bacterial infection, and/or a t least 1 , 5 fold decrease in transcription levels of one or more genes that decrease in expression level upon bacterial infection.
In some embodiments, the present teachings include an anti-viral drug for use m a method of treatment of a disease caused by a virus, characterized in that the patient has at least a L5 fold, increase in transcription levels of one or more genes that increase in expression level upon viral infection, and/or at least a 1.5 fold decrease in transcription levels of one or more genes that decrease in expression level upon viral infection.
In some embodiments, the present teachings include an antibiotic for use in a method of treatment of a disease caused by bacteria, characterized in that the patient has been selected to have at least, a i .5 fold increase in transcription levels of one or more genes that increase in expression level upon bacterial infection, and/or at least a 1.5 fold decrease in transcription levels of one or more genes that decrease in expression level upon bacterial infection.
In some embodiments, the present teaching include an anti-viral drug for use in a method of treatment of a. disease caused by a virus, characterized i that the patient has been selected to have at least at least a 1.5 fold increase in transcription levels of one or more genes that increase in expressio level upon viral infection, and/or at least, a 1.5 fold decrease in transcription levels of one or more genes that decrease in expression level upon viral infection. I» some embodiments, the present teachings include an antibiotic for use in a method of treatment of a disease caused by bacteria, characterised in that the method comprises a determination, of whether the patient has at least a 1.5 fold increase in transcription, levels of one or more genes that increase in. expression level upon bacterial infection, and/or at. least a 1.5 fold decrease in transcription levels of one or more genes that decrease in expression level upo bacterial infection.
In some embodiments* the present teachings include an. anti-viral drug for use in a method of treatment of a disease caused by a virus, characterized in that the method comprises a determination of whether the patient has at least a L5 fold increase in
transcription levels of one or more genes that increase in expression level upon viral infection, and/or a t least a. 1.5 fold decrease in transcription levels of one or more genes that decrease in expression level, upon viral infection.
In some embodiments, the present, teachings include an antibiotic for use in a method of treatment of Fever without an Apparent Source (FWS) in a subject, characterized in that the subject has in a sample such as a blood sample at least, a 1.5 fold increase in expression level compared to a control level of at least one endogenous gene selected from the group consisting of RHOU, GNAI 3, PPP1 S 2A, RHOT1, FCER1G, LYN, RALB, GNAQ, MARCKS, TGM2, ARHGEF1 1 , MYL12A, EP300, MYL9, CREB5, FCGR2A, GNG10, GNGI 1, C1QR NOD2, TLR2, TLRi , R.NASEL, C5A.RE TLR4, MYD88, P1 3CB, C ARE TLR0. CASPE TLR5. NERC4, TER8. ILIB, PPP1R.E ZYX, YL12 , VA.SP, UGA2B, ITGB5, VCL, 1TGB3, M YLK, ASAPE ITGAM, ITGAX. FCGR3B, SIGLEC9, FCERIG, j AK-2, CASPS and a combination thereof.
In some embodiments, the present, teachings include an antibiotic for use in a method of treatment of Fever without an Apparent. Source (FWS) in a subject, characterized in that the subject has in a sample such as a blood sample at least a .1.5 fold increase in expression ievel compared to a control level of at least one endogenous gene selected from the group consisting of FY , CD247, EITPR3, CD3, ZAF70, PLCG L PRK.CFL LCK, EAT, PRK.CQ, ΓΓΚ, ITGB7, TSPAN4, KXRDE IR2DE3, KIR2DE4, K1R3DL3, IR3DLE HCST, C.D247, NCR3 and a combinatio thereof.
In some embodiments, the present teachings include an antibiotic for use in a method of treatment of Fever without an Apparent Source (FWS) in a subject, characterized in that the subject has in a blood sample at least a 1.5 fold decrease in expression level compared to a control level of Annexin A3 gene. I» some embodiments, the present teachings include an anti-viral drug for use in a method, of treaiment of Fever with an Apparent Source (FWS) in a subject. In various aspects, the F WS can be characterized in thai the subject can have in a sample such as a blood sample at least a 1.5 ibid increase in. expression level compared to a control level of at least one endogenous gene selected from the group consisting of IF 127, ISO 15, Ol OF, IF1T3, ITGAM, ITGAX and a combination thereof.
In some embodiments* the present teachings include an anti-viral drug for use in a method of treatment of an adenoviral infection in a subject, characterized in that, the subject can have in a sample such as a blood sam le an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of ATM., PRKCFL PRKCQ, C IQB, CI QC, IRF7, OAS3, OAS1 , OAS2, AK2, ΕΪΡ2, IPIHl , DDX58, NOD2, T.LR5, NLRC4, TLR.8, C3AR.1 , ILI B, TLR1 , TLR4, TLR.6, MYD88, CASP!, IFIT3, 1FI35 BCL2, MED 14. IFNGR2, IFNARl , IRFE BAX, IRF9, PSMB8, IFFF I , JAK2, STAT2, TAPE IKBKB, IKBKAP, ΚΪΑΑ 2 1, TRAF6, TNF. TBK1 , TANK, 1RF , NFKBIA, IRP7, IS 15, ADAR, ZBPE IFIT2, FOS, LY96, 1XR5, IRAK3, FI RS, EIF2AK2, CD 14, MAPK14, STAT4, HS.572649, CCR7, CD40LG, LIB, HLA-DOA, CI1EB5, FCGR3B, FCGR2A, ILI N, LTBR, TYROBP, FCE S G, FCORI A, FCGRI B and a combination thereof.
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of an adenoviral infection in subject, characterized In that the subject can hav e in a sample such as a blood sample an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting ofClQB, CIQC, IRF7, OAS3, OAS I, OAS2, AK2, EIF2, IFIHE DDX58, NOD2, TLR5, NLRC4, TLR8, C3ARL ILI B, TLR l , TLR.4, TLR.6, MYD88, CASPL IFIT3, IFI35, BCL2, IFNARI, !RFL BAX, 1RF9, PS B8, IFITMi , JAK2, STAT2, TAPE TRAF6, TNF, TB3K K TANK, IRF9, NFKBIA, IRF7, 1SG15 ADAR, ZBP l, IFIT2, FOS, LY96, TLR5, 1RAK3, TLR8, E1F2A 2, GDI 4, MAPK14, CREB5, FCGR3B,
FCGR2A, ILI N, LTBR, TYROBP, FCERIG, FCGRI A, FCGRIB and a combination thereof.
In some embodiments, the present teachings include an anti-viral drug for use in a method of tTeatraeni of an adenoviral infection i a subject, characterized in that the subject can have in. a sample such as a blood sample an aberrant level of expression, at least a 1 ,5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of ATM, PRKCFL PRKCQ, MED 14, IFNGR2, IKBKB. IKBKAP, IAA127 L STAT4, HS.572649, CCR7, CD40LG, LT.B, HLA-DOA and a combination thereof and a combination thereof,
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of an enteroviral infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant level of expression at least a 1 , 5 fold greater compared to a control level of at least one endogenous gene selected from the group consisting of NL C4, TLR8, TLR5, NOD2, RNASBL, TLR2, TLRl, CS RI , YD88, TLR6, CASP1, IL1B, C iQB, IRF7, OAS1, OAS 3, BFF2AK2, DDX58, IFIM, OAS2, If IT!, IFST3, IFB5, M 1 , TAF1, STAT2, IFITM1 , STAT I , JAK2, IRF7, ISG15, T2, DMX58, ZBP1, FOS, LY96, MAP2K3, MAPKI4, CASP5 and a combination thereof
'.In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of an enteroviral infection in a subject, characterized in that the subject can have in a sample such as a. blood sample an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting ofCCLS, PR CH, TLR5, TLR2, NOD2, TLR8, TLR4, T'LR6, CI OA, MYD88, RIPK2, IL! B, C3AR.1, CASPL CI QB. CI QC OAS 1., 1RF7, OAS3, EIF2AK2, TLR7, OAS2, DDX58, IFIHL IPIT3, l.FLT'S , SOCSI , MX'L FB5, IFIT L TAPL STAT2, JAK2, STAT1 , TYROBP, CD86, IL1B, CA.SP5, CCL2, TLR9, TRA.F6, IRAK4, CHUI , CD 1.4, MYD88, TLRl , FOS, LY 6 and a combination thereof.
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of an enteroviral intection in a subject, characterized in that the subject can ha ve in a sample such as a blood sample an aberrant level of expression, at least a 1.5 fold greater compared to a control level of at least one endogenous gene selected from the group consisting of NLRC4, TLR8, TLR5, MOD2, RNASEL, TLR2, TLRl , C5.AR1 , MYD88, TLR.6, CASPL ILI B, C1QB, IRF7, OAS 1 , OAS 3, E1F2AK2, DDX58, IF 11:11, OA.S2, ΪΡΓΠ, 1P3T3, IF135, MX1, TAPL STAT2, IFITML STAT L JA 2, IRF7, ISO 15, IFIT2, DFIX58, ZBPf, FOS, LY96", MAP2K3, MAPK14, CASP5 and a combination thereof
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of an enteroviral infection in a subject, characterized, in that the subject has in a blood sample an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of CCL5, PRKCH, TLR5, TLR2, NOD2, TLRB, TLR4, TLR6, C1QA, MYD8S, RIPR2, ILI B, C3ARL CASPL CIQB, CiQC, OAS I, IRF7, OAS3, EIF2AK.2, TLR7, OAS2, DDX58, IFffl L ΙΠΤ3, IFIT1 , SOCSI , M.X 1 , IFD5, IFITML TAP STAT2, IAK2, STAT1 , TY.ROBP, CD86, IL1 B; CASP5, CC.LL TLR9, TRAFi IRA 4, CHUK, CD14, MYD88, TXR1 , FOS, LY96 and a combination thereof
In some embodiments, the present teachings include an anti-viral drug for use i a method of treatmen t of an 'HHV-6 infection in a subject, characterized in thai the subject can have in a sample such as a blood sample an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting ojf CCL5, FRKCH, TLR9 and a combination thereof
In some embodiments, the present teachings include an anti-viral drug for use hi a method of treatment of an BHV-6 infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant level of expression of at least 1 ,5 fold increase compared, to a control level of al least one endogenous gene selected from, the group consisting ofCD N!C MT2A, SPATS2L, 1RF7, ZBP1 , OA .2, OAS 1, 1SG1 S, IF.16, HERC5, OAS3, RSAD2, OAS!,, IF1T3, OTOF and IF 27.
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of an. HHV-6 infection in a subject, characterized in that the subject can have in a sample such, as a blood sample an aberrant level of expression of at least 1 .5 fold decrease compared to a control level of at least one endogenous gene selected from the group consisting of YH9 and ARAP3.
In some embodiments, the present teachings include an anti-viral drug' for use in a method, of treatment of an HHV-6 infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant le vel of expression of at least 1.5 fold increase compared to a. control level of at least one endogenous gene selected irom the group consisting of CD N1C, MT2A, SPATS2L, IRF7, ZBPL OAS2, OAS1 , ISG15, TFI6,
HERC5, OAS3, RSAD2, OASL IFIT3, OTOF and IFI27 and at least 1.5 fold decrease compared to a control level of at least one endogenous gene selected from the group consisting of MYH9 and ARAP3,
In some embodiments, the present teachings include an antibiotic for use in a method of treatment of a bacterial infection in a subject, characterized in that a sample such as blood sample of the subject can. have at least a 1 ,5 fold decrease in expression, of at least one endogenous gene selected from the group consisting of CD N1C, MT2A, SPATS2L, IRF7, ZBPI , OAS2, OAS I, 1SG15, FJ.6, HERC5, OAS3, RSAD2, OASL 1FIT3, OTOF and FI27. In some embodiments, the present teachings include an antibiotic for use in a method of trea tment of a. bacterial infection in a subject, characterized in thai a sample such as a blood sample of the subject can have at least a 1.5 fold increase in expression of at least one endogenous gene selected from the group consisting of MYH9, ARAP3 o a combination thereof.
In some embodiments, the present teachings include an antibiotic for use in a method of treatment of a bacterial infection in a subject, characterized in that a sample such a a blood sample of the subjec t can have at least a 1.5 fold decrease in expression of at least one endogenous gene selected from the group consisting of CD NiC, MT2A, SPATS2L, I F7, ZBPL OAS2, OAS I, ISO 15, 1-16, MERCS, OAS3, RSAD2, OASL, lFil'3, OTOF a d 27, and at least 1.5 fold increase in expression of at least one endogenous gene selected from the group consisting of YB.9, ARAP3 and a combination thereof.
In some embodiments, the present teachings include an antibiotic for use in a method of treatment of a bacterial infection, characterized, in that, a sample such as a. blood sample of the subject can have an aberrant level of expression at least 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group
consisting of FY , CD247, EUPR3, CD3, ZAP70, PLCG L PRKCFL LC , LAX, PRK.CQ, IXK, RHOU, GNA 1 , PPP1R12A, RHOX1, FCER IG, FYN, RALB, GNAQ, ARCK.S. TGM2, ARIIGEFI 1 , MYLI 2A, EP300, MYL9, CREB5, FCGR2A, GNG !O, GNGl J ,
CIQB, NOD2, TLR2, T.LRL RNASEL, C5AR I, TLR4, MYDSS, PIK3CB, C3AR1 , TLR6, CASPl, TLR5, NLRC , TLR8, IF I B, JXGB7, TSPAN4, PPPI 1 , ZYX, VASP, fFGA2B, IXGB5, VCL, ITGB3, MYLK, ASA P L 3TGAM, ITGAX, LRD1, K1R2DL3, KIR2DL4, KIR3DL3, KJR3DLI. HCST, CD247, NCR3, FCGR3B. SIGLEC9. FCE iG, JAK2, CASP5 and a combination thereof.
In some embodiments, the present teachings Include an antibiotic for use in a method of treatment of a bacterial infection, characterized in that a sample such as a blood sample of the subject can have an aberrant level of expression that is at least a F5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group
consisting of RHOU, GNAI3, PPP1 12A, RHOT5 , FCBR!G, LYN, RALB, GNAQ, MAR.CKS, TGM2, ARHGEFI 1 , MYL1.2A, EP300, MYL9, CRFB5, FCGR2A, GNG10, GNGl 1, C IQB, NOD2, TLR2, TLRI, RNASEL, C5ARF TLR4, MYD88, PI 3CB, C3AR1, XLR6, CASPl . TLR5, NLRC4, TLRS, ILIB, PPPI RJ , ZYX. MYL12A, VASP, ITGA2B, 1TGB5, VCL, LXGB3, MYLK, ASAP 1 , 1TGAM, IXGAX, FCGR3B, SIGLEC9, FCERIG, JAK2, CASP5 and a combination thereof.
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of an enieroviral infection in a subject, characterized, in that the subject can have in a sample such as a blood sample an aberrant level of expression that is at least, a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of adenoviral, infection is selected from the group consisting of NERC4, TER8, TLR5, OD2, RNASEL, TLR2, TLRl , C5AR1, MYD88, TER6, CASPE IL1B, CI QB, IRF7, OASE OAS3, E1F2AK2, DDX58. IF1HE OAS2, 1FITE IFIT3, 1FDS, MX 1, TAP i , STAT2, !FITMl, STATU J 2, 5RF7, ISO 15, 1FIT2, DBX58, ZBPI , POS, EY96, MAP2K3, MAPK14, CASP5 and a combination thereof
In some embodiments, the present teachings include an anti-viral dru for use in a method of treatment of an enteroviral infection in a subiect, characterized in that the subject can have in a sample such as a blood sample -an aberrant level of expression that is at least a 1,5 fold increase compared to a control level of at least one endogenous gene selected from the group consisting of NERC4. TER8. TLR5. NOD2, RNASEL, TLR2, TLRl , C5AR1 , MYD88, TLR6, CASPE I LI B, CIQB, IRF7, OAS I, OAS3, EIF2AK2, DDX5S, IFIH E OAS2, IF1TL 1FIT3, IFI35. MX I , TAPE STAT2, IFIT i , STATE JAK2, IRF7, ISG15, 3ΕΠ2, DHX58, ZBFE FOS, EY96, MAP2K3, MAP 14, CASP5 and a combination thereof
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of an HBV- infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant, level of expression that is at least a 1.5 fold decrease compared to a control level of at least one endogenous aene selected from the group consisting of CC1.5, PR CH and. TLR9.
In some embodiments, the present teachings include an anti-viral drug for use in a meth od of treatment of a viral infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant level of expression that is at least a 1.5 fold increase of at least one endogenous gene selected from the group consisting of CDKNIC, MT2A, SPATS2L, IRF7, ZBPE OAS2, OAS E ISGI5, IPI6, HERC5, OAS3, RSAD2, OASE, IFFT3, OTOF and IFI27, compared to a bacterium-infected control level.
In some embodiments, the present, teachings include an anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in. that the subject has in a sample such as a blood sample an aberrant level of expression that is at. least, a E5 fold decrease of at least one endogenous gene selected from the group consisting of MYH9 and ARAP3, compared to a bacterium-infected control level.
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in thai the subject can have in a sample such as a blood sample an aberrant level of expression that i at least a 1 ,5 fold increase of at least one endogenous gene selected from the group consisting of BAK E 1FNGRI , STAT2, IF135, MX I, OAS! , IFIT1 and IFIT3, compared to a bacterium-infected cont.ro! level.
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in. that the subject can have in a sample such, as a blood sample a aberrant level of expression thai is at least a 1.5 fold decrease of at least one endogenous gene selected from the group consisting of RRAS, ACTR2, CK2, PDC3CB, MAP2K4, ΠΌΑ.2Β, ITGB3, MYLK, MYL9, II'GBS, GNG 1 L ZYX, ITGAX and ITGAM, compared to a bacteriura-infected control level
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant level of expression that is at least a 1.5 fold increase of at least one endogenous gene selected from the group consisting of IFNGRl , IFNGR2, MT2A, SPATS2L, OAS2, OAS I, iSG l S, IFI6, iFITL HERC5, OAS3, RSAD2, OASL, OTOF and 1FI27, compared to a bacterium-infected control level.
In some embodiments, the present teachings include an anti-viral drug for use in a method of trea tment of a viral infection in a subject, characterized in that the subject can have in a blood sample an aberrant level of expression that is at least a 1 .5 fold decrease of at least one endogenous gene selected from the group consisting of OSBPL8, VHL, ACTR2, MAP2K4, P13A1 , PROS1 , ITGB3, MYL9, ITGA2B, !TGBS, GNG1 1 , EP300, ZYX, ARAP3. AGER, ITGAX, SORLI and KJTAM, compared to a bacteriura-infected control .level.
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in that the subject can have in a sample such as a blood sample an aberrant level of expression that is at least a 1.5 fold- increase of at least one endogenous gene selected from the group consisting of 1FI27, ISO 15, OTOF and IFIT3, compared to a bacterium-infected control level
In some embodiments, the present teachings include an anti-viral, drug for use hi a method of treatment of febrile HHV-6 in a subject, characterized i that the subject can be selected to have in a sample such, as a blood sample expression that is at least a .1.5 fold increase of at least one endogenous gene selected from the group consisting of IF127, IFi'O, and a combination thereof compared to an afebrile HHV-6 control level or a febrile control level.
In some embodiments, the present teachings include an anti-viral drug for use in a method of treatment of febrile HHV-6 in a subject, characterized in that the subject can be selected to have in. a sample such as a blood sample expression that is at least a IS fold increase of at least one endogenous gene selected from the group consisting of IF127, JSCS 15, and a combination thereof compared to an afebrile adenovirus control level or a febrile control level
In some embodiments, the present teachings include an anti-viral drug for use in a meihod of treatment of febrile enterovirus in subject, characterized in that the subject can be selected to have in a sample such as a blood sample expression that is at least a 1.5 fold increase of at least one endogenous gene selected Irom the group consisting of IFI27, ISO 15, and a combination thereof, compared to an afebrile enterovirus control level, or a febrile control level.
In some embodiments, the present teachings include an. antibiotic for use in a method of treatment of bacterial infection in a subject, characterized in that the subject can be selected to have in a blood sample expression tha is at least a 1.5 fold increase of at least one endogenou gene selected from the group consisting of ITGAM and ITGAX, compared to an afebrile control level or a viral-infected control level,
BRIEF DESCRIPTION OF THE DRAWINGS
For some drawings that were originally in color, a composite of 3 color channels (red, green and blue), along with individual color channels, are shown.
FIG. 1 illustrates identificatio of virus- and bacterial response-specific probes, FIG. 1 A illustrates Venn diagram showing identification of virus- and bacterial response-specific probes. FIG. I B illustrates heat maps of gene expression of viral response-specific probes, FIG, IC illustrates heat maps of gene expression, of bacterial response-specific probes. Each row represents a gene with expression value normalized to the mean of the afebrile virus- negative control group and each column represents one individual, in original color, red represents up-regulation and blue represents down-regulation.
FIG. 2 illustrates blood leukocyte transcriptional profiles of febrile and afebrile HFiV-6- positive children compared t those of afebrile virus-negative children, FIG. 2A illustrates a clustering of probe sets. FIG. 2B illustrates principal component analysis of differentially expressed genes, with each oval representing one child. FIG. 2C, FIG 2D, FIG. 21-3, and FIG. 2F illustrate clustering of differentially expressed genes of FIG. 2A according to expression intensity In ingenuity® canonical pathways. Each row represents a gene whose expression value is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column, represents one individual. In original color, red represents up- regulation and blue represents down -regulation. FIG, 3 illustrates probes specific for individual viruses and for bacteria. Each .row represents a probe and each column displays probes for one febrile subject positive for the indicated virus or with acute bacterial infection.
FIG- 4 illustrates classifier probes discriminating febrile children positive ibr viruses from febril children with acute bacterial infections, FIG, 4A illustrates 1581. gene-based classifiers, FIG. 4B illustrates 18 gene-based, classifiers, FIG, 4C illustrates 22 pathway-based classifiers, and FIG. 41) illustrates 33 classifiers selected from, gene-based and pathway-based classifier sets. Class is designated by green (viral) or blue (bacterial) letters: A, adenovirus; B. Bacteria; E, enterovirus; II, HH V-6, Classification based on patients* white blood cell ( WBC) count is shown beneath each heat map. The upper strip shows classification based on age-specific normal values and the lower strip shows classification based on a cutoff of 15,000 cells per cii mm.
FIG. 5 illustrates blood transcriptional profiles of febrile adenovirus-positive children and profiles of afebrile adenovirus-positive children and afebrile controls. FIG. 5A illustrates clustering of probe sets. FIG, SB illustrates a principal component, analysis of differentially expressed genes, with each oval representing one child. FIG. SC. FIG. 51), FIG. 5E, FIG. 51% and FIG. 5G illustrate clustering of differentially expressed genes from FIG . 5 A according to expression intensity in Ingenuity® canonical pathways. Each row represents a gene with expression value that is normalized to the mean of the afebrile virus-negative- control group. Gene names are listed to the left. Each column represents one individual. In original color, red represents up-regulation, and blue represents down-regulation.
FIG, 6 illustrates blood transcriptional profiles of enterovirus-positive febrile children and virus-negative afebrile children. FIG. 6 A. illustrates clustering of probe sets. FIG. 6B illustrates a principal component analysts of differentially expressed genes, with each oval representing one child, FIG. 6C, FIG 61), FIG. 6Έ, FIG. 6F, and FI.G. 6G illustrate clustering of differentially expressed genes in FIG. 6A according to expression intensity in Ingenuity® canonical pathways- Each row represents a gene with expression value that is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column, represents one individual. In original color, red represents up-reguHuiou, and blue represents down-regulation,
FIG. 7 illustrates blood transcriptional profiles of febrile children with acute bacterial infections and profiles of virus negative afebrile children. FICi. 7A illustrates clustering of probe sets. FIG, 7B illustrates a principal component analysis of differentially expressed genes, with each oval representing one child. FIG. 7C, FIG 7D, FIG. 7E, FIG. 7F, and FIG. 7G llustrate clustering of differentially expressed genes in FIG. 7 A according to expression intensity in ingenuity® canonical pathways. Bach row represents a gene with expression value that is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column represents one individual. In original color, red represents up-regulation, and blue represents down-regulation.
FIG. 8A and FIG. 8.8 illustrate selected up- and down-regulated Ingemnty 5 canonical pathways identified for febrile children positive for adenovirus, HHV-6, or enterovirus and febrile children with acute bacterial infections. The pathways are arranged, in ascending order by average P value of four infections for a pathway.
FIG. 9 illustrates quantile -normalized raw signal intensity of the classifier probes in febrile children. FIG. 9A illustrates 18 classifiers identified from. 260 viral- and 1 ,321 bacterial response-specific probes. FIG. 9B illustrates 22 classifiers identified from 34 genes in the Ingenuity® IFN signaling pathway and 205 genes in the Ingenuity® integral signaling pathway. FIG. C illustrates 33 classifiers identified from using both gene-level and pathway- based approaches. FIG. 9D illustrates relative expression data of nine classifier genes. In FIG. 9 A - 9D, each dot represents one sample.
FIG, 10 illust.ra.tes validation of three sets of classifier probes discriminating virus-positive febrile children from febrile children with acute bacterial infection using three independent cohorts of subjects, FIG . 10A illustrates validation with a set of 785 probes overlapped across three datasets with 1 ,58.1 virus- and bacterial response-specific probes. FIG. 10B illustrates validation with gene-based classifiers. FIG. IOC illustrates validation with pathway-based classifiers, FIG. iOD illustrates validation with hybrid gene- and pathway-based classifiers. For FIG. 10A ···· FIG. 10D: Patient groups are indicated by stripes at the top of the heat map, hi original color, predicted class is labeled with green for viral or blue for bacterial infection. Gene names in green in original signify genes selected from the viral-specific gene set (or the IFN signaling pathway genes), and gene names in blue in original represent genes selected from the bacterial-specific gene set (or the integrin signaling pathway genes). Heat map rows are gene probes, whereas columns are individual subjects.
FIG. 1 1 illustrates samples from febrile subjects with confirmed viral/bacterial infection. FIG, Ϊ 1 A illustrates correlation of transcriptional changes and leukocyte subpopulations in the subjects, FIG, 1 IB illustrates an expression, pattern of corresponding 4,716 probe sets in cluster format. Rows represent genes and columns represent individual samples, Dashed lines indicate the lowest, values of correlation coefficients significant (adjusted P < 0.05) for each parameter. DETAILED DESCRIPTION
Abbreviations
ANOVA Analysis of variance
BAX Bel-2 -associated X protein
FDR. False discovery rate
FWS Fever Without an Apparent Source
GAPDH Glyceraidehyde 3-pbosphate dehydrogenase
GEO Gene expression omnibus
I ll lV-6 Human herpesvirus 6
1FN interferon
LAMP Loop-medialed isothermal amplification
Lymph Lymphocyte
Mono Monocyte
MRSA Methiciiiin-resisian Staphylococcus aureus
MSS A Methicil I i n-sensiti ve Staphylococcus aureus
Neul Neutrophil
PAM Prediction Analysis of Microarrays
PCR Polymerase chain reaction
REN RNA integrit number
RNA-seq RN A Sequencing, Whole Transcriptome Shotgun Sequencing
Tm Melting temperature
QC Quality control
WBC White blood ceil
AACt Comparative CT Method
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinar skill in the art to which the present teachings belong. Singleton el «/., Dictionary of Microbiolog and Molecular Biology 2nd ed J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry
Reactions, Mechanisms and Structure 4th e ,, John Wiley & Sons (New York, N.Y. 1 92), provide a person of skill in the art with general guides to many of the terms used, in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present teachings. The present teachings are in no way limited to the methods and materials described. For purposes of the present teachings, the follo wing terms are defined below.
As used herein, "microarray" refers to an ordered arrangement of hybridkab!e array elements including but not limited to polynucleotide probes, on a substrate.
As used herein, "biological sample" is any sampling of cells, tissues, or bodily fluids containing cells. Examples of biological samples include, but are not limited to, peripheral blood mononuclear cells, a nasopharyngeal, sample, a urine sample, a blood sample, a lumbar puncture sample, a bodily fluid, a biopsy sample, a tissue sample and a combination thereof,
As used herein, "blood" ncludes whole blood, plasma, and serum.
As used herein,, "aberrant expression levels" includes an increase or decrease of gene expression compared to baseline, or compared to a appropriate comparison group.
Methods and compositions described herein utilize laboratory techniques well known to skilled artisans. Such techniques can be found in laboratory manuals such as Sambrook, .1», et at.. Molecular Cloning: A Laboratory Manual, 3rd e l. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001: Specter, D. L, et a!.. Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998; Methods in
EnzyrnoSogy (Academic Press, Inc.); Handbook of Experimental Immunology, 4th edition (D. M. Weir & C. Blaekwel!, eds., Blaekwell Science Inc., 1 87); Current. Protocols in Molecular Biology (F. M. Ausubel et. al> eds., 1987); and PGR; The Polymerase Chain Reaction, (M'uliis tw/., eds., 1 94). Methods of administration of pharmaceuticals and dosage regimes, can be determined according to standard principles of pharmacology well known skilled, artisans, using methods provided b standard reference texts such as
Remington: the Science and Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1 95): Hantaan, J.G., et al., Goodman & Oilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1.996; and Rowe, R..C, et al,, Handbook of Pharmaceutical Excipients, Fourth Edition, Pharmaceutical Press, 2003 , As used in the present description and any appended claims, the singular forms "a", "an" and " he" are .intended to include the plural forms as well, unless the context indicates otherwise.
Methods of gene expression profiling can include methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides. Methods for quantification of niRNA expression in a sample include northern blottin and in situ hyb idization (Parker & Barnes, Methods in Molecular Biology 106:247-283 ( 1.999)); RNAse protection assays (Hod, Y., Biotechniques 1 92, 1.3:852-854); and. reverse transcription- polymerase chain reaction (RT-PCR) (Weis et at., Trends in Genetics 8:263-264 (1992)). Alternatively, antibodies can be employed that can recognize specific duplexes, including DMA duplexes, RNA duplexes, and DNA-R . hybrid duplexes or O A-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSSK For example, RT-PCR can be used to compare mRNA levels in different sample populations, in normal and infected tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between, closely related niRNAs, and or to analyze RNA structure,
A, first step for an RT-PCR. analysis can be extraction and/or isolation of mRNA from a sample. In some embodiments, starting materia! can be total RNA isolated from a human PBMC. RNA can be isolated from a variety of PBMCs, such as, without limitation, a lymphocyte, a leukocyte, a monocyte or a macrophage.
Methods for mRN A extraction are well known i the art and are disclosed in standard textbooks of molecular biology, including Ausubel et «/,, Current Protocols of Molecular Biology, John Wiley and Sons ( 1 97), RNA isolation can be performed using a purification kit, buffer set and proteas from commercial manufacturers, such as Qiagen, according to manufacturer's instructions. For example, total RNA from cells in culture can be isolated usina Qia¾en RNeasv® mini-columns. Other commercially available RNA isolation kits include MasterPureTM Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), and Paraffin Block RNA Isolation Kit (Ambion, inc.). RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test), RNA prepared from a biological sample can be isolated, for example, by cesium chloride densit gradient cenirifiigation.
A. first step in gene expression profiling by RT-PCR can be reverse transcription of an RNA template into cDNA, followed, by amplification in a PGR reaction. For example, extracted RNA can. be reverse-transcribed using a GeneAmp RNA PGR kit (Perkin Elmer, Walthani, MA USA), following manufacturer's instructions. cDNA can then be used as template in a subsequent PGR amplification and quantitative analysis using, for example, a TaqMao® (Life Technologies, Inc., Grand island, NY) assay.
TaqMan.® RT-PCR (life Technologies, Carlsbad, CA, USA) can be performed using commercially available equipment, such as, for example, an Α.ΒΪ PRISM 7700TM Sequence Detection SystemTM (Perkiti-Elmer-Applied Biosystems, Foster City, Calif, USA), or Jighteye!er (Roche Molecular BioehemicaLs, Mannheim, Germany), RT-PCR can be performed using an internal standard such as mRNA for glyceraldehyde-3-phosphate- dehydrogenase (GAPDH) and/or β ---aetin as a control (see, e.g.. Held et Genome
Research 6: 986-994, 19%).
In some embodiments, PCR primers and probes can be designed based upon intron sequences present in the gene to be amplified, in such aspects, a first step in primer/probe design can be the delineation of intron sequences within, the genes. This can be accomplished using publicly available software, such as the DMA BLAST software (Kent, W. I. Genome Res, 12(4): 656-664, 2002). Subsequent steps can include the following established methods of PCR primer and probe design.
in some configurations, in order to avoid .non-specific signals, repetitive sequences within nitrons can be masked when, designing primers and probes. This can be accomplished by using software such as the Repeat Masker program available on-line through the Baylor College ofMedicine, This, program can be used to screen DNA sequences against a library of repetitive elements and returns a. query sequence i which the repetitive elements are masked. Masked intron sequences can then be used to design primer and probe sequences using a commercially or otherwise publicly available primer/probe design package, such as Primer Express (Applied Bsosysteras); MGB assay-by-deslgn (Applied Biosystems); Pomer3 (Rozen, and Skaietsky, H.J., (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetx S, Misener S (eds) Bioin ormatics Methods and
Protocols; Methods in Molecular Biology. Humana Press, Totowa, N.J., pp 365-386);
Methods Mol. Biol. 1 2, 365-386, 2000).
Factors considered in PCR primer design can include primer length, melting temperature (Tra), G/C content, specificity, complementary primer sequences, and 3 -end sequence- PCR primers can be, but are not limited to, 1.7-30 bases in length, and contain about 20% from 0% to 80%, or about 80% G - C bases, such as, for example, about 50%, from 50 to 60%, or about 60% G+C bases, in various configurations, Tm's can be between 50 and 80° C„ e.g. about 50 to 70° C.
Further guidelines for PCR primer and probe design can be found in various published sources, e.g. Dieffenbach, C. W. et L, "General Concepts for PCR Primer Design" in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 533-3.55; innls and Geifaod, "Optimization. of PCRs" in: PCR Protocols, A Guide to Methods and Applications* C C Press, London, 1994, pp. 5-1 1 ; and Plasterer, T. N, Priroerselect: Primer and probe design. Methods Mol. Biol. 70:520-527 ( 1 97), the entire disclosures of which are hereby expressl y incorporated by reference. In some embodiments, differential gene expression can be identified, or confirmed usi ng a microarray technique. In these methods, polynucleotide sequences of interest (including cDNAs and oligonucleotides) can be plated, or arrayed, on a .microchip substrate. The arrayed sequences can be hybridized with specific DNA probes from cells or tissues of interest. RNA can be isolated from a variety of biological sources, In. an embodiment of the microarray technique, PCR-amplified inserts of cDNA clones can be applied to a substrate in a dense array. Mieroarrayed genes, immobilized on a microchip, can be suitable for hybridization under stringent conditions.
In some embodiments, fluorescently labeled cDNA probes can be generated through incorporation of fluorescent nucleotides by reverse transcription: of RNA extracted from tissues of interest Labeled cDNA probes applied to a chip can. hybridize with, specificity to loci of DNA on the array. Afte washing to remove non-specificaily bound probes, a chip can be scanned by confocal laser microscopy or by another detection method, such as a CCD (charge coupled device) camera. Quantification of hybridization of each arrayed element, can allow for assessment of corresponding mRNA.
In some configurations, dual color fluorescence can be used. With dual color fluorescence, separately labeled cDNA probes can be generated from two sources of RNA and can be hybridized pairwise to an array. The relative abundance of transcripts from the two sources corresponding to each specified gene can be determined simultaneously. In various configurations, the miniaturized scale of the hybridization can afford a convenient and rapid evaluation of an expression pattern for large numbers of genes, in various configurations, such methods can have sensitivity to detect rare transcripts, which are expressed at fewer than 1000, fewer than 100, or fewer than 10 copies per cell, hi various configurations, such methods can detect at least approximately two-fold differences in expression, levels (Schena ei al., Froc. Natl. Acad. Sci. USA 93(2): 106-149 (1996)). In various configurations, microarray analysis can. be performed by commercially available equipment, following manufacturer's protocols, -such as by using AfTymetrk GenChip technology, or Ineyte's microarray technology.
Non-limiting representative protocols for profiling gene expression including mRNA isolation, purification, primer extension and amplification are given in various published journal articles (for example; Godf ey, T.E., t tL 2000, J, Molec, Diagnostics 2: 84-91 ; Spech.t, K, ei « ., 2001 , Am. J. Pathol. 1.58: 41.9-29),. RNA can be extracted, and protein, and DNA can be removed.. Alter analysis of the RNA concentration, RNA repair and/or amplification steps can be included, and RNA can be reverse transcribed using gene specific promoters followed by RT-PG1. Data can be analyzed to identify a treatment option(s) available to the patient on th basis of the characteristic gene expression pattern identified in the examined biological sample.
EXAMPLES
The present teachings including descriptions provided in the Examples mat are not intended to limit the scope of any claim or aspect. Unless specifically presented in the past tense, an example can. be a prophetic or an actual example. The .following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art, in light of the present disclosure, will appreciate thai many changes can be made in. the specific embodiments that axe disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.
Example 1
This example illustrates detection of viruses in young children with Fever Without an Apparent Source (FWS),
Subjects were drawn from a study of children between 2 to 36 months of age with Fever Without an Apparent Source ( Table 1 ) and afebrile children having ambulatory surgery who were recruited at St Louis Children's Hospital as described previously (Colvin, J.M., et at. 2012, Pediatrics 1.30(6):e 1455- 1462). The febrile and afebrile groups were similar with respect to age, gender, and season of recruitment, but differed with respect to race, with more African-American children in the "febrile group (57% vs. 13%). Patients were enrolled according to Institutional Re view Board- ppr ved protocol. The study was approved by the Washington University Human Research Protection Office. Each subject was tested for m ultiple viruses in blood and nasopharyngeal samples using panels of virus-specific PC assay as described (Colvin, J.M., el al. 2012, Pediatrics 130(6):el455-1462), Subjects were selected for the study of gene expression profiles if they were positive for a single virus in one or both samples. The viruses included, were adenovirus, HHV-6, enterovirus, and rkinoviras. Also included were- subjects- who had a definite bacterial infection (bacteremia, urinary tract infection, skin and soft tissue infection, bone or joint infection) and whose samples were negati ve for viruses tested, as well as a group of subjects selected from the afebrile control group whose samples were also negative for viruses tested (Colvin, I.M., et ah 2012, Pediatrics i30(6}:el455-1462). Statistical analysis did not reveal race as a confounding variable.
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9284 Cotttr So Me 1 W h ite Example 2
This example illustrates preparation of biological samples, including RNA from blood samples.
In these investigations, whole blood and nasopharyngeal samples were collected for virus-specific PCR and high-throughput sequencing. Additionally, a blood sample was collected in a Tempus™ Blood RNA Tube (Applied Biosysiems, Carlsbad, California) and stored at >80°C. for subsequent gene expression, analysis.
Total RNA was isolated from whole blood collected in. Tempus™ Blood RNA tubes (Applied Biosystems, Carlsbad, CA) according to the manufacturer^ instructions. RNA quality was determined by gel-chip image (showing 28S, 18S and 5S bands) and RNA integrity number (RIN, generally a >? RIN indicates good quality RNA) using an Agilent 21.00 Bioanalyzer (Agilent, Palo A l to, CA), All but 3 of the RN A preparations had RIN scores > 7.0. Total R A concentration wa obtained from an absorbance ratio at 260.nm and 280nm using a NanoDrop ND-100 spectrometry instrument (NanoDrop inc., Wilmington, DE).
Example 3
This example illustrates gene expression micfoarray assays.
I these investigations, gene expression microarray analyses were conducted on blood samples from 35 febrile children positive for adenovirus, human herpesvirus 6 (iTllV-6), or enterovirus infection or with acute bacterial infection, and 22 afebrile controls. Gene expression, microarray assays were carried out at the Genome Technology Access Cente in Washington University in St. Louis. RNA transcripts were amplified by T7 linear
amplif cation with the Il!umina 3'IVT Direct. Hybridization Assay Kit (Illumina inc., San Diego, CA), and biotin-labeled cRNA targets were hybridized to the Illumina Human-HT12 v4 Expression BeadChips (>47,000 probes), which were scanned on an Illumina BeadArray Reader. Scanned images were quaniitated by 'illumina Beadscan software (v3). Quantified data were imported into illumina GenomeStudio software (versio 201. 1.1) to generate expression profiles and to make data quality assessments. These data have been deposited into GEO database at the National Center for Biotechnology Information (GEO ID:
GSE40396).
Example 4
This example illustrates microarray data analysis including differential expression analysis, pathway analysis, and identification of classifier genes, class prediction and unsupervised hierarchical, clustering. In these investigations, host transcriptional response was analyzed at the level of up- and down-regulation of individual genes and of functional gene pathways. Differences were detected of up - and down -regulation of individual genes in transcriptional profiles in febrile children positive for any of the three viruses and with acute bacterial infection. Several approaches to developing panels of probes were used. A panel of individual gene probes was developed based on the strength of statistical association with a type of infection. A panel was developed of genes from two pathways that differentially activated: the Interferon Signaling Pathway, activated in febrile virus-positive children, and the integrisi Signaling Pathway, activated in children with, acute bacterial infection, A hybrid approach was additionally used, in which genes were selected, from each of the gene-based and pathway- based approaches. The large set of gene-based probes (1581) and each of the three
"shrunken" approaches that used between 18 and 33 probe functioned for classification purposes.
For microarray data analysis, expression profiles were generated in the GenomeStudio and imported into Partek Genome Suite (version 6.6, Partek Inc., Saint Louis, MO). Data, quality was assessed across individual samples using principal component analysis and hierarchical clustering analysi that, could identif specific sample clusters thai were associated with nonexperimemal factors (such as chip effect, age. gender, and array QC metrics). Five of 70 samples that had the lowest number of detectable probes were identified as outliers and eliminated without farther analysis. A total of * 26,300 probes that had been detected (detection p-vaksc <0.01) in at least one of the 70 samples were kept in downstream statistical analysis and quantSIe-normaiS ed for differential expression analysis.
For differential expression analysis, analysis of variance (ANOVA) was performed in Partek Genome Suite for genes with differential expression in viral and bacterial infection and afebrile controls, with adjustment for batch effect (hybridization date and individual chips). P- values from the ANOVA were corrected for false discovery rate (FDR, or q-vahie). With the exception of symptomatic enterovirus infection, analyses were conducted with p- value <0.05 and FDR at 5%,
For pathwa analysis, pathways that were roost activated for each virus and for bacteria! infection were identified from the mgem»ty# Pathway Analysis (Ingenuity® Systems, Redwood City, CA) library of canonical pathways. The significance of the association between the data set and the canonical pathway was assessed in. two ways: 1 ) The ratio of the number of up- and down-regulated probes from the da ta set included in the pathway divided by the total number of probes that were included in the canonical pathway; 2) statistical evaluation using Fisher's exact test of the probability that the association 'between, the genes in the dataset and the canonical pathway is explained by chance alone.
Identification of classifier genes, class prediction and unsupervised hierarchical clustering: The -nearest neighbor classification algorithm embedded in the Prediction Analysis of Mfcroarrays (PAM) tool, was used to identify classifier genes presenting the highest capability to discriminate the two classes of bacteria! and viral infection (TibshiranL IL, et ah 2002, "Diagnosis of multiple cancer types by shrunken eentroids of gene expression " Proc. Natl Acad Set USA 99(! 0):6567-65?2), With iO-nearest neighbors and 10-fold cross-validation, P.AM calculates misclassificatio error rate in the training set of data for each of the two classes according to varying threshold (a unique statistical parameter), A threshold is chosen when the misclassifreation error is minimized for both classes to define a subset of probes from the entire training data set, designated as classifier probes. "These classifier probes were used for class prediction on testing data sets. Hierarchical clustering was used with the complete linkage- lgorithm to evaluate the accuracy of classification.
Transcriptional profiles of virus-positive febrile children and febrile children with acute bacterial infection differed from those of afebrile virus-positive and afebrile virus- negative children,
Mieroarray data was analyzed from lllumina Human HT12 Bead-Chips comprising -47,300 probes hybridized, with UNA samples extracted from whole blood specimens from 22 febrile children (8 positi e for huma herpesvirus 6 [MHV-6], 8 positive for adenovirus and 6 positive for enterovirus, and 8 with acute bacterial infection). The same mieroarray assay was performed, on blood samples from 35 afebrile children (2 positive for HHV-6, 3 positive for adenovirus, 8 positive for riiinovirus, and 22 virus-negative control children).
By using a strategy based on intersecting variou probe sets as described previously (Chaiissabel D, et L 2005, Annals NY Acad. Sci. 1062; 146-154), probes sets were intersected that were significantly up- or down-regulated for each of the virus-positive groups and the febrile acute bacterial infection group compared to afebrile virus-negative, control children (FIG. 1A).
FIG. 1 illustrates identification of virus- and bacterial response-specific probes for distinguishing virus-positive febrile children and febrile children with acute bacterial infection from virus-negative afebrile children. FIG. 1 A illustrates a Ven diagram showing identification of virus- and bacterial response-specific probes. Sets of probes differentially expressed in febrile children positi ve for 1 or more of the 3 viruses compared to vims- negative afebrile control children were intersected and 1.6 1 "panvirus" probes were identified and intersected with the set of probes thai were differentially expressed in children with febrile acute bacterial infection compared to virus-negative afebrile control children. From this analysis, 413 virus-specific and 1939 bacterial response-specific probes were identified, including probes specific for individual viruses. FIG. IB and FIG. I C illustrate heat-maps showing gene expression based on 260 viral response-specific probes ( FIG. 1 B) and 1321 bacterial response-specific probes (FIG. IC) in children with febrile and afebrile viral and bacterial infections and afebrile virus-negative control children. These probes were selected in (he same manner as the 413 vims -specific and 1939 bacterial response-specific probes described above except that for this selection, with the exclusion of 2 of 22 virus- negative controls. Probes that were not annotated in GenBank Build36 (National Center for Biotechnology Information) were also excluded. F+. febrile, afebrile. Each row represents a gene whose expression value is normalized to the mean of the afebrile virus-negative control group and each column represents one individual. Red in original represents up- regulation and blue represents down-regulation.
Using this method 260 probes with significant up- or down-regulation specifically In virus-positive febrile children and 1321 probes with significant up- or down-regulation specifically in children with febrile acute bacterial infection were identified. Analysis of the 260 viral probes revealed overlap in gene expression profiles for febrile children who were positive for adenovirus, HHV-6, or enterovirus infection, which were very different from the profiles of most afebrile children (FIG. .1 B). Profiles of virus-positive and virus-negative afebrile children were indistinguishable. Analysis using the 1321 bacteria! probes displayed similar patterns of gene expression for most of the children with, fever and acute bacterial infection that differed from those of the other groups, with a few exceptions (FIG. IC).
The extensive differences in gene expression, profiles between febrile and afebrile children were further analyzed for each virus and for acute bacterial infection by principal component analy sis and by analysis of genes grouped into Ingenuity® canonical pathways (www.ingenuiiy.com). Results for HHV-6 are shown in FIG. 2, and results for adenovirus, enterovirus, and acute bacterial infection are available as FIGS. 5, 6, and 7,
FIG. 2 illustrates blood leukocyte transcriptional profiles of febrile and afebrile HHV- 6-positive children compared to those of afebrile virus -negative children.
I these investigations, microatray analysis was conducted o RN A extracted from blood samples of 1 children positive for HRV6 (8 febrile, 2 afebrile), and 22 afebrile vims- negative children. FIG. 2A illustrates clustering of probe sets with a statistically significant > 2-fold difference between HHV~6-positive febrile children and afebrile virus-negative control children (FDR 5%). FIG. 2B illustrates a principal component analysis of differentially expressed genes, with each oval representing one child. FIGS. 2C-2F illustrates clustering of differentially expressed genes in FIG. 2A according to expression intensity in 4 Ingenuity® canonical pathways. Each row represents a gene whose expression value is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column represents one individual. Red represents up-regulaiion and blue represents down- regulation.
FIG, 5 illustrates blood transcriptional profiles of febrile adenovirus-positive children as different from the profiles of healthy children and afebrile children with adeivoviros infections.
In these investigations, microarray analysis was conducted on RNA extracted from blood samples of 1 1 children with confirmed adenovirus infection (8 febrile and 3 afebrile children) and 22 afebrile virus-negative children. FIG. 5A illustrates clustering of probe sets with a statistically significant and greater than two-fold difference between adenovirus- positive febrile children and virus-negative afebrile controls (false discovery rate (FDR) at 5%). FIG. 5 B illustrates a principal component analysis of differentially expressed genes, with each oval representing one child. FIGS, 5C-5G illustrate clustering of differentially expressed genes from FIG. 5A according to expression intensity in five Ingenuity® canonical pathways. Each row represents a gene with expressio value that is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column represents one individual. Red represents up-regtsSation, and blue represents down-regulation.
FIG, 6 illustrates blood transcriptional profiles of enterovirus-positive febrile children, as different from the profiles of virus-negative afebrile children.
In these investigations, microarray analysis was conducted on RNA extracted from whole-blood samples of 6 enterovirus-positive febrile and 22 virus-negative afebrile children. FIG. 6A illustrates ciustering of probe sets with, a statistically significant greater than twofold difference between enterovirus-positive febrile children and virus-negative afebrile controls (P < 0.05, FDR at 20%). FIG, 6B illustrates a principal component analysis of differentially expressed genes, with each oval representing one child. FIGS. 6C-6G illustrates clustering of differentially expressed genes in FIG. 6A according to expression intensi ty in five Ingenuity® canonical pathways. Each row represents a gene with expression value that is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column represents one individual. Red represents up-regulation, and blue represents down-regul at ion. FIG. 7 illustrates blood transcriptional profiles of febrile children with acute bacterial infections as different from the profiles of vims-negative afebrile children.
In these investigations, raicroarray analysis was conducted on RNA extracted from whole-blood samples of 8 febrile children with confirmed bacterial infectio and 22 virus- negative afebrile children. FIG. 7 A illustrates clustering of probe sets with a statistically significant greater than two-fold difference between febrile children with acute bacterial infection and virus-negative afebrile controls (FDR. at 5%). FIG. 7B illustrates a principal component analysis of differentially expressed genes, with each oval representing one child. FIGS. 7C-7G illustrate clustering of differentially expressed genes from FIG. 7 A. according to expression intensity in five Ingenuity® canonical pathways. Each row represents a gene with an expression value that is normalized to the mean of the afebrile virus-negative control group. Gene names are listed to the left. Each column represents one individual Red represents np-regulation, and blue represents down-regulai on.
Pathways with the most significant transcriptional changes for children with each of the three viral infections and with acute bacterial infection are shown hi FIG. 8 A and FIG. 8B.
FIG. 8A aftd FIG. 8B illustrate selected significantly up- and down-regulated
Ingenuity® canonical pathways identified for febrile children positive for adenovirus, human herpesvirus 6 (Hf IV-6), or enterovirus and febrile children with acute bacterial infections. The pathways were arranged in ascending order by average P value of four infections for a pathwa (i.e., the most significantly up- or down-regulated pathway is at the top).
Comparison of individual probes between HBV-6~positive febrile children with virus- negative afebrile children yielded 3467 probes with significant transcriptional changes, including 798 probes with- 2-fold or greater changes (606 up- and 1 2 down-regulated. FIG. 2A), A principal component analysis of the transcriptional profiles confirmed clear differences between the febrile and afebrile HHV-6-positive children (FIG. 2B).
A. sample from, the' one child in the virus-negative afebrile control group with a transcriptional pattern in the beat-maps shown in FIG. IB and FIG. 1 C similar to those with febrile infection was classified with the febrile infections in. the principal component analysis. An up-regulated gene was IFI27/1SG 12 A, Analysis of transcriptional pat h ways showed some pathways with up-regulation of many component genes as illustrated in FIG, 2C-FIG. 2F.
The gene expression profile of adenoviras-positive febrile children is illustrated in FIG. 5, Statistical comparison of transcriptional profiles between adeno virus-positive febrile children and virus-negative afebrile children showed 5604 probes with significant transcriptional changes including 847 with a 2-fold or greater Increase (576) or decrease (271 ) in expression level Principal component analysis confirmed the differences between the febrile and afebrile adenovirus-posi ive children, IFI27/ISG 12A was up-regulated. FIG. 8 displays the pathways with the significant transcriptional changes.
Comparison of transcriptional profiles of enteroviras-positive febrile children and virus-negative afebrile control children yielded 4184 probes with significant changes as shown in FIG. 6. The magnitude of these transcriptional changes was generally less than those for adenovirus and HHV-6, and therefore FDR was set at 20% to maximize the possibility of detecting either up- or down-regulated genes. This yielded 678 probes with 2~ fold or greater transcriptional change (559 up- and 1 19 down-regulated). 1FI27/ISG12A was an up-regulated gene. The pathways with significant transcriptional changes in febrile enterovirus-positive children arc shown in FIG. 8,
Statistical comparison of microarray data between febrile children with acute bacterial infection and virus-negative afebrile control children yielded 1234 probes with 2-fold or greater change with either up-regulation (850) or down-regulation (384) (FIG. 7). An up- regulated gene was Aiinexhi A3 ( 14.6-fold).
Transcriptional pathways were differentially activated in febrile children with viral and bacterial infections. Some Ingenuity® canonical pathways had significant transcriptional changes in febrile children positive for one of the three viruses (HHV-6, adenovirus, enterovirus) or with acute bacterial infection. Pathways thai were activated in each of the four infection groups were Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses, TREMl Signaling, and Toll-like Receptor Signaling. Some genes .trom the Natural Killer Cell Signaling pathway were down-regulated in each of the four infection groups. The Interferon Signaling Pathway and the Activation of interferon Regulatory Factors by
Cytosolie Pattern Recognition Receptors Pathway were more activated in febrile virus- positive children compared to febrile children with acute bacterial infection. In contrast, genes in the hitegrin Signaling Pathway were activated in bacterial infection. Transcriptional changes in each of these pathways are displayed in FIG. 8,
Unique sets of genes were associated with speci fic viral and bacterial infections in febrile children, In some examples, signaling pathways were similarly activated among the different viruses tested in this study, but with significant variations in the expression level of many individual genes. 2078 probes were identified with, significant transcriptional changes uniquely present in adenovirus-positive febrile children, 464 uniquely present in HHV-6- pos ve febrile children, 594 uniquely present m enterovirus-positive febrile children and 1939 uniquely present in febrile children with acute bacterial, infection (FIG. I A). Using the shrunken ceiitroid algorithm (Tibshirani, «, ei at. 2002, Proc. Nat '!, Acad. Sci. U S A
99( i0):6567-6572), subsets of specific gene probes for each of the individual viruses and for acute bacterial infection were identified (Table 2). These virus-specific transcriptional profiles and the profile specific for acute bacterial infection are shown in FIG. 3.
Table 2. Individual virus- and bacteria-specific: profile gene probes with strongest effects
Adenovirus «s. <smvct# HWV-Svs. a y'.roi Srstefovi us as. contrai 8».tef¾s ¾. <0>tf: vi
Fol Αο3:···.Λΐ Abutted fififss; symboi cosoos^ Chang* P v*iu<>-
SETN NM..020 1S.2 •5.62» 0,007 1,?<1S 0.223 1554 8.475 1,887 0. ;?2
OU¾!4 ftfv ; 6 5S 5.776 0.0?? 1.028 o.s5¾ ) .570 3.338 3.150 0 in NNLO00233.1 2.130 ο,ο» --1030 0.622 -1,223 0,530 ' A''-- 0.433
OPSSKAS NM..004255.2 -1.701 0,0.37 0.4S7 102? 8,072 Ϊ7Ϊ.5 0 .53 :
TSPYi.2 NMJB22W.1 ■■1.632 0,002 -1.113 043 ? --■,15? 3.380 -■1,250 0 00 nm -1,625 6.808 -1143 0.4 IS 1052 0.SS9 •.345 0.120 ecus 2.S¾ 0.104 12.-SSS 0.!¾» 2,003 0,3 ?9 .059 0 505 cu 2 i >8 o.m 3.752 2,221 3.284 -1,187 0.44S i-RRCSO MM J 78 5;. 3 1.212 0.334 3.42$ owe .ί- .' 0.430 -1.074 0.470
VP528 »t.Q S28&2 -- 1 sm 0.434 -1625 QMS - 1,348 0,040 5.0-54 0.423
Wv1..00S00S.2 i.iaa 0.5¾3 - 1.562 ΰ,ϋΖΰ i. 03 CMS! . 0.576
NM.133*86. ? ; 3S !>.3*s 1349 0. .18 3.1 SO ti.om i.S4« 0.2.78
HASH NM.CC532 •342 0.212 -1 103 0567 2,152 5,190' 0.443
RM..182*73.1 1.236 - .·· «; 5, CMS 0,610 2.057 0.059 1,30! 0.226
KLO 1 1375 0.433 - 1.142 0,198 1.999 0.083 1.307 0.245
51¾¾½!. :AC4 1.2SS 0.103 -105S 0.S01 945 mm -1.04S 0.487.
WM..000313. S 1.16?. 0.294 - 10J.9 0.S2S 1.255 2.473 0,001
SC¾g 1C1 -•1.017 0.522 1.152 0.492 1230 3.485 2.483 oxm
ASAF3 ί¾ .022·183 5 tm 0.X3S -1104 0.545 1.26?. 3.440 2,201 (1667
«M..'>^2« .2 - T.-S'i 0. 560 - i .281 0.244 1.884 0.595 2.050 ϋ.ϋΰ4
G2MH ΝΜ..0 423.3 "1.328 0.100 -1.733 0.234 ■-2.0SS 3,280 -5.258 0M3
KlfUOU *S ..0S4S11.3 -1.016 O S2*5 -3.092 03501 -1.S42 0,548 --3.Ϊ89 0.007
KSJDW NMJ¾22:SS,S 1.165- 0.3S4 1.052 0.615 - ! T!OS ¾405 -2.645 twin
KifiSDU Ε¾Μ013288 5 - ; ¾ s o.s:¾ -1101 0.577 1 J--; 0.433 -2.572 0.008
Mi X fi?v1..C05352. :.!0?' 0 414 1 .291 0.393 1.170 05Λ5 - 1.787
053PL3 !M.,":-:«¾«.5S. S --1.814 0.ϋ ·¾ 1.187 0.32S - !,101 3.388 -1,787
Table 2: These probes were had significant up- or down -regulation in children •positive for only one virus or with acute bacterial infection using an adjusted P value of 0.05. Gene transcription in children positive for only one virus and children with acute bacterial infection were each compared with gene transcription in afebrile virus-negative control, children. Candidate genes were derived using the shrunken centroid algorithm procedure in the Prediction of Mi.eroar.ray Analysis tool from Stanford Universit (littp; /www- stat.stanibrd.edu/--tibs SAM ). Probes were sorted within each virus/bacteria grou by descending fold change (when up-regulated) or ascending fold change (when down- regulated). Bold and italic fonts indicate genes that at adjusted F value < 0.05.
FIG. 3 illustrates probes specific for individual viruses and for bacteria, in these investigations, viral -spec fic and bacterial response-specific probes were subjected to the shrunken centroid algorithm individually for each of the 4 pathogen groups to find a non- limiting reduced number of probes with ability t differentiate among pathogen groups. Each row represents a probe and each, column displays probes for one febrile child positive for the indicated vims or with acute bacterial infection.
Classifier probes were identified to distinguish viral and bacterial infections in febrile children with validation on independent daiasets. Host transcriptional profiles unique to either viral or bacterial, infection were characterized to assist in making this clinical discrimination, individual-gene-based and pathway-based approaches for selecting probes were compared. For the gene-based approach* a "master set" was used, including the 1581. (260 viral- and 3321 bacterial-specific) probes described above and a limited subset of 38 of the 1581 selected using the shrunken centroid -algorithm. For the pathway-based approach, the shrunken centroid algorithm was used to select 22 probes from the Interferon Signaling Pathway (selectively activated in virus-positive febrile children) and the Integrm Signaling Pathway (selectively activated in febrile children with acute bacterial infection). The hybrid approach used 33 probes selected using the shrunken centroid algorithm from the master set and from the Interferon Signaling and Integrin Signaling Pathways, 9 classifiers were selected from the 3 sets of classifiers described above and validated by RT-qPCR. High correlation in expression level was found for 9 classifier genes between RT-qPCR and microarray results (FIG. 9, Table 3).
Table 3, Correlation in expression level between RT-qPCR and microarray results
Classifier Pearson correlation of coefficient P value
§R2? 0,722 2.17E-07
IRT1 0.903 3.55E-15
1SG15 0.775 S.79E-Q9
ITGAM 0.925 4.29E-17
1TGAX 0,813 3.1 1
ITG85 0.B86 6.86E-14
OASL 0.937 1.66E-18
OTOf 0.927 2.5SE-17
PROS1 0.705 5.47E-07
Classification of cases was carried out using unsupervised hierarchical clustering and the K-nearest neighbor algorithm. The "true class" of each case was based on virus-specific PCRs and bacterial cultures as previously described (Coivin JM, t ah 2012, Pediatrics i30(6):e 1455- 1462). The classifications derived from the use of probes selected by each approach are shown in FIG. 4.
FIG. 4 illustrates classifier probes that discriminate febrile children positive for viruses from febrile children with acute bacterial infections. FIG. 4A illustrates 1581 gene- based classifiers. FIG, 4B illustrates 1.8 gene-based classifiers. FIG. 4C illustrates 22 pathway-based classifiers. FIG. 41) illustrates 33 classifiers selected from gene-based and pathway-based classifier sets. In each panel, patients are displayed as. columns and probes as rows. Gene symbols are shown in blue for bacterial infection-specific genes and in green for viral, infection-specific genes. Expression values presented in the heat map were normalized to the mean, of the afebrile virus-negative control cases. Hierarchical clustering was used to classify patients into two groups with the majority of cases classified as either viral (green tree branch) or bacterial (blue tree branch). Classification as predicted using the K-nearest neighbor algorithm is shown as a bar above each heat map, with green showing classification as viral and blue showing classification as bacterial. True class was determined by virus- specific PG and bacterial cultures, and is designated by green (viral) or blue (bacterial) letters: A, adenovirus; B, Bacteria; E, enterovirus; II, IJHV-6. Classification based on patients' white blood cell (WBC) count is shown beneath each heat map. The upper strip shows classification based on age-specific norma! values and the lower strip shows
classification based, on a cutoff of 15,000 cells per cu mm.
The signal Intensity of the probes is shown in FIG. 9, and classification performance of each set is summarized in Table 4, Correct classificatio based on hierarchical clustering ranged from 77-90% and 83-90% based on die ~nearest neighbor method.
Table 4. Prediction Accuracy of Four Sets of Classifier Probes
Oista set * Ati 1SS1 vfius-sf>s¾ifi£ S ciassiffers itom 1S81 22 dsssftiws from 33 dassifisr* sele ted using probes withtsut s«SsritoB bacter)a«pectfk probes * imegiin pathway < 1 osthwisy »d ap r ach *·!
¾i: S is s 127 12δ 9*2¾>
Figure imgf000044_0001
* Number of cases in data set
1" 785 of 15.81 gene-based probes were identified in the amilo data sets
i 182 of 23 pathway-based genes were identified In the Ramilo data sets
I Number (percent) of classified eases 1 Ramilo O, el al, (2007) Blood 109(5):2066~2077
FIG. 9 illustrates quanlile-no.rmalized raw signal intensity of classifier probes in 30 febrile children (22 virus-positive children and S children with acute bacterial, infection) in our study. FIG. 9 A illustrates 18 -classifiers identified from 260 viral- arid 1,321 bacterial response-specific probes. FIG. B illustrates 22 classifiers identified from 34 genes in the ingenuity® IFN signaling pathway and 205 genes in th Jngenuity® (Ingenuity Systems, inc., Redwood City, CA) integral signaling pathway, FIG- 9C illustrates 33 classifiers identified •from using both gene-level and pathway-based approaches. FIG. 9D illustrates relative expression da ta of nine classifier genes, generated in quantita tive RT-PCR (RT-qPCR).
validation assays for 29 of 30 febrile children (one RNA sample with BHV-6 infection was not available for the assays). The expression level was calculated using ΔΔΟί method, and normalized to endogenous reference GAPDl-L Each dot represents one sample.
Independent datasets from Ramilo O, et al 2007, Blood 109(5): 2066-2077 were used to test the clinical validity and robustness of classifier probes for distinguishing viral and bacterial infection. The validation data included three different cohorts analyzed using three different microarray platforms, each of which differed from ours, 95% accuracy was achieved in distinguishing viral and bacterial infection using the 158 i probes and. 88- 1% accuracy in using the other three sets of probes (Table 4, FIG- 1.0).
E mple 5
This example illustrates RT-qPCR validation assays.
For RT-qPGR validation assays, primers and probes of assay-on-demand were purchased from life Technologies (Applied Biosystems, Forster City, CA), and master mix was from Quanta Biosciences (Gaithersburg, MD) tor .reverse transcription and quantitative PCR (RT-qPCR) assays. The assays were carried out in triplicate on an ABS 7500 real-time PGR. instrument following manufacturer's protocols. Assays had >80% PCR efficiency and <15% coefficient of variance in triplicate reactions.
Performance of probe sets was validated using three previously published microarray data sets (Ramilo O, et al. 2007, Blood 1 9{5):2066-2077). Comparisons were made across different microarray platforms. Selected probes achieved classifications that were 88-95% concordant with the classifications of the validation sets.
FIG. 1.0 illustrates validation of three sets of classifier probes discriminating virus- positive febrile children from febrile children, with acute bacterial infection using three independent cohorts of patients. The 1 -sample validation set included. 18 subjects with influenza A, 29 subjects with Escherichia eoU, 31 subjects with Staphylococcus aureus, and J 3 subjects with Streptococcus pneumoniae. This set was profiled with the AiTyrneirix ( Santa Clara, California USA) Human Genome UI33A Array platform. The 22-sample validation set included seven children with influenza A, three children with influenza B, six children with 5. aureus, and six children with & pneumonia. This validation set was profiled with the Afiy etrix Human Genome U i 33 Plus 2.0 Array platform. The 24-sample validation set included 5 subjects with influenza A, 3 subjects with influenza B, 13 subjects with '. aureus, and 3 subjects with S. pneumoniae, and it was profiled with the illoraina Sentrix Human-6 Expression BeadChip platform. Overall prediction, accuracy was 95% (130/137),
88%(120/137), 88% ( 121 137), and 91 % (124/137) with FIG. 1 OA a full set of 785 probes overlapped across all three datasets with 1 ,581 viral- and bacterial response-specific probes, FIG. I DS gene-based classifiers (n « 1 8), FIG. IOC pathway-based classifiers (n « 22), and FIG, !OD hybrid gene- and pathway-based classifiers (n ~ 33), respectively. Patient groups are indicated by colored stripes at the top of the heat. map. True class indicates status determined by virus-specific PGR assays and bacterial cultures, and it was assigned to these cases in Ramilo, O.. et al (2007), J#o ^ I O9(5):2066~2O77). Predicted class was determined by prediction made with the classifier probes, and it is labeled with green for viral or blue for bacterial infection.. Gene names in green signify genes selected from the viral -specific gene set (or the IFN signaling pathway genes), and gene names in blue represent genes selected from the bacterial-specific gene set (or the integral signaling pathway genes). Expression values presented in the heat-maps were normalized to the mean of cases with bacterial infection within each datasef. Heat-map rows are gene probes, whereas columns are
Individual, subjects.
Example 6
This example illustrates utilizing the Pearson test to determine correlation between gene expression profiles and white blood cell counts / differentials.
In these investigations, the Pearson test was used to find significant correlations of differentially expressed genes with white blood cell and differential counts in the 30 febrile cases. The differentia! expression was defined with p<0.05 and fold-change >l .5 in comparisons between febrile groups and healthy controls. The original p- value of the correlation coefficient was adjusted by multiple test correction and the adjusted, p-value was set at 0.05 for significance. Age-specific normal, values for white blood cell count used by the clinical laboratory at. St. Louis Children's Hospital: <1 week: 5.0-30.0 /cu mm; 1 week-! month: 5.0-20.0 K/cu mm; 1 month-2 years: 6.0-17.5 K/cu mm; 2-6 years: 5,0-15,5 K/cu nm; 6-12 years: 4,5-13.5 K/cu mm; >12 years: 3,8-9,8 K/cu ram.
A .Pearson correlation test was performed for 4716 probes that were different from virus-negative controls in febrile children. The total white blood cell count was not. associated with, gene expression level, but expression of several clusters of genes was significantly associated with neutrophil, lymphocyte, or monocyte counts, suggesting that those clusters of genes might be acti vated in specific types of cells (FIG. 1.1).
FIG, 1 1 illustrates correlation of transcriptional changes and leukocyte subpopulations in febrile young children. In. these investigations, whole-blood samples were collected from 30 febrile children with .confirmed viral/bacterial infection. Probe sets with at least a 1.5-fold change in level of expression over virus-negative afebrile controls are shown. The expression pattern of the corresponding 4,716 probe sets is displayed in hierarchical cluster format, where rows represent genes and columns represent individual samples. Correlation coefficients were calculated between the expression level of each probe set and white blood ceil, counts (total, neutrophil, lymphocyte, bands, and monocyte counts) across 30 patients. The correlation values are plotted as moving averages of 50 probe sets (along the vertical axis). Dashed lines indicate the lowest values of correlation coefficients significant (adjusted P < 0.05) for each parameter.
Host transcriptional profile results were more accurate compared, to traditional white blood cell count for discriminating bacterial from viral infection. Previous studies indicate that white blood cell count is an inadequate tool for distinguishing between viral and bacterial infection, distinction ofte used to determine whether or not to treat the patient with antibiotics (Rudinsky, S.L., etal 2009, Aca Emerg Med 16(7):585~590 and E&rz, A.M., et al. 2006, Pedkiir Infect Dis J 25(4):293~300). Classificatio based on transcriptional profiles was compared with classification based on white blood cell count, using a cutoff of
15,000/ nr' as recommended by the American Academy of Pediatrics in their guideline for the management of febrile children 0-36 months of age (Bara.ff, Li., el al. 1.993, .Ann. Emerg. Med. 22(7): 1 198-1.210), A different set of cutoffs was also analyzed based o age-specific normal values for white blood cell count used by the clinical laboratory at St Louis
Children's Hospital. The classifier gene probes were more accurate for distinguishing bacterial and viral infection than either of the white blood cell criteria as illustrated in FIG. 4. Example 7
This example illustrates diagnosis of a. symptomatic adenovirus infec tion in a subject. In this example, a patient exhibits symptoms that initially appear attributable to a viral or bacterial infection. A health practitioner obtains a whole blood sample* a nasopharyngeal sample, and a blood sample in a Tempus™ Blood RNA Tube (Applied Biosystems, Carlsbad, California) from the subject. A. health practitioner orders a pathogen culture and a. serum antibody detection test. The health practitioner add tionally orders a test to determine expression levels of 260 viral response-specific endogenous genes and 1321 bacterial response-specific endogenous genes by a microarray hybridization assay. The pathogen culture is negative and the serum antibody test is positive for an adenovirus. The microarray hybridization assay reveals an increase in expression of 165 of the 260 viral, response-specific endogenous genes, and a decrease of 1200 of the 1321 bacterial response-specific endogenous genes. The patient is diagnosed with a symptomatic adenovirus infection.
Example 8
This example illustrates diagnosis of a symptomatic bacterial infection in a subject.
In this example, a patient exhibits symptoms that initially appear attributable to a viral or bacterial infection. A health practitioner obtains a. whole blood sample, a nasopharyngeal sample, and a blood sample in a Tempus™ Blood RNA Tube (Applied Biosystems, Carlsbad, California) from the subject. A health practitioner orders a pathogen culture and a serum antigen detection test. The health practitioner additionally orders a test to determine expression levels of 260 viral response-specific endogenous genes and 13 1 bacterial response-specific endogenous genes by a microarray hybridization assay. The pathogen culture is positive forS. aureus and the se um antibody test is negative. The microarray hybridization assay reveals a decrease in expression of 206 of the 260 viral response-specific endogenous genes and an increase in 1 145 of the 1321 bacterial response-specific
endogenous genes. The patient is diagnosed with a symptomatic bacterial infection, specifically S. aureus infection.
Example 9
This example illustrates a method of diagnosis of Fever Without an Apparent Source in a subject.
A patient exhibits symptoms of Fever Without an Apparent Source. A health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in a Tempus™ Blood NA Tube (Applied Biosystems. Carlsbad, California) from the subject. A health, practitioner orders a pathogen culture and a serum antibody detection test The health practitioner additionally orders a. test to determine expression levels of the following endogenous genes; OSBPL8, VEIL, ACTR2, MAP2 , F13A1 , PROS! , ITGB3, YL- , ITGA2B, ITGB5, GNGI 1 , EP300, ZYX, ARAP3, AGER, ITGAX, S .RLI, IFNGRL
EFNGR2, ITGAM, MT2A, SPATS2L, OAS 2, OAS I , IS.GJ S, IFI6, IF!TL BERC5, OAS3, RSAD2, OASL, OTOF and IFI27 by microarray hybridization assay. The pathogen culture is negative and the serum antibody test is positive for an enterovirus. The 'microarray hybridization assay reveals an increase in expression of IF GR1, JFNGR2, MT2A,
SPATS2L* OAS2, OAS!, ISO 15, IF16, ΙΡΓΠ , HERC5, OAS3, RSA.D2, OASL, OTOF and DF127 and a decrease in expression ofOSBPLS, VBL, A. F 2, MAP2K Fl 3 A.I , PROSE ITGB3, MYL9, ITGA2B, FTGB5, GNGI 1, EP3O0, ZYX, ARAP3, AGER, ITGAX, SORLl and IGTAM. The patient is diagnosed with a symptomatic enterovirus infection.
Example 10
This example illustrates a method of diagnosis of Fever Without an Apparent Source in. a subject,
A patient exhibi ts symptoms of Fever Wi thout an Apparent Source, A health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a 'blood sample in a Tempus™ Blood RNA Tube (Applied Biosysiems, Carlsbad, California) from the subject A health practitioner orders a pathogen culture and a serum antibody detection test. The health practitioner additionally orders a test to determine expression levels of the following endogenous aenes: BARE ERAS. A.CTR2, NCK2. PIK3CB, MAP2K4. ITGA2B, HGB3. MYLK, MYL9, ΓΓΟΒ5, GNGI I, ZYX, ITGAX, !FNGRl , ITGAM, STAT2, IF135, MX1 , OASL I'FITI and ΙΡΓΓ3 by microarray hybridization assay. The pathogen culture is indefinite and the serum antibody test is negative. The microarray hybridization assay reveals an increase in expression of BA ! , IPNGRE STAT2, IFB5, MX. I, OAS! , IFiTIand IFIT3 and a decrease in expression of RRAS, ACTR2, NCK2, PIK3CB, MA.P2K4, ITGA2B, 3TGB3, MYL , MYL9, ITGB5, GNGI I, ZYX, ITGAX and ITGAM. The patient is preliminarily diagnosed with an unknown and symptomatic viral, infection.
Example 1 1
This example illustrates a method of distinguishing a viral-caused infection from a bacterial-cause infection or a combination thereof.
A patient exhibits symptoms that initially appear attributable to a viral or bacterial infection, A health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in a Tempus™ Blood RNA Tube (Applied Biosysiems, Carlsbad, California) from, the subject A health practitioner orders a pathogen, culture and a serum pathogen antigen detection test. The health practitioner additionally orders a test to determine expression levels of the following endogenous genes: MYB9, ARAP3, CD IC, T2A, SPATS2L, IRF7, ZBP1, OAS2, OAS] , ISO 15, IFI6, HERC5, OAS3, RSAD2, OAS.L. IF1T3, OTOF and IFI27 by microarray hybridization assay. The pathogen culture is negative and the serum antigen test is positive for adenovirus. The raicroarray hybridization assay reveals an increase in expression of CD 1C, T2A, SPATS2L, IRF7, ZBP1, OAS2, OAS!, IS015, 1F16, HERC5, OAS3, RSAD2, OAST, IFFB, O TOF and 1F127 and a decrease n expression of MYH9 and ARAP3, The patient is diagnosed with a symptomatic viral infection.
Example 12
This example illustrates a method of distinguishing a viral-caused infection from a bacterial-cause infection or a combination thereof.
A patient, exhibits symptoms that initially appear attributable to a viral or bacterial infection. A health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in a Ter pus™ Blood UNA Tube (Applied Biosystenis, Carlsbad, California) from the subject. A health practitioner orders a pathogen culture and a serum pathogen antigen detection test. The health practitioner additionally orders a test to determine expression levels of the following endogenous genes: MYH9. AR.AP3, CD N 1C, MT2A, SPATS2L, IRF7, ZBP1 , O S2, OAS L 1SG15, IF16, HER.C5, OAS3. I SAD2, OA.SL, IFIT3, OTOF and 1F127 by niicroarray hybridization assay, The pathogen culture is positive for E.coli and the serum antigen, test is negati ve. The microarray hybridization assay reveals a decrease in expression of CD N1C, MT2A, SPATS2L, 1RF7, ZBPI , OAS2, OAST ISG15, IF16, HERC5, OA.S3, R.SAD2, DASL, 1FIT3, OTOF and IF127 and an increase in expression of ΜΥΉ9 and ARAP3. The patient is diagnosed with a symptomatic bacterial infection. Example 13
This example .illustrates a . method of diagnosis of a pathogen-associated disease.
A patient, exhibits symptoms that initially appear attributable to a viral or bacterial infection. A. health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in a Tenipus™ Blood RNA Tube (Applied Biosyste is, Carlsbad, California) from the subject. A health practitioner orders a pathoge culture and a serum pathogen antigen detection test. A health practitioner orders at least one test to determine expression levels of the following endogenous genes: IFI27, iSG! 5, OTOF, IFIT3, 1TGAM, and ITGAX by raicroarray hybridization assay. The pathogen culture is negative and the serum antigen test is positive for adenovirus. The raicroarray hybridizatio assay reveals a decrease in expression ofJTGAM, and ITGAX and an increase in expression of IF.I27, ISGI S, OTOF, and IF1T3. The patient is diagnosed with a symptomatic adenoviru infection. Example 1.4
This example illustrates a method of diagnosis of a pathogen-associated disease.
A patient exhibits symptoms that initially appear attributable to a viral or bacterial infection. A health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in. a Tempos™ Blood RNA Tube (Applied Biosystems, Carlsbad, California) from the subject. A health practitioner orders a pathogen culture and a serum, pathogen antigen detection lest. A health practitioner orders ai least one test to determine expression levels of the following endogenous genes: 1FI27, ISG15, OTOF, 1F1T3, ITGAM, and ITGAX by mic.roar.ray hybridization assay. The pathoge culture is positive for B.coli and the serum antigen test is negative- The microarray hybridization assay reveals an increase m expression of ITGAM, and ITGAX and a decrease in expression of IFI27, ISGl 5, OTOF, and ΠΤΤ3, The patient is diagnosed with a symptomatic bacterial infection.
Example 15
This example illustrates a method of diagnosis of a viral pathogen-associated disease.
A patient exhibits symptoms thai initially appear attributable to a viral or bacterial infection. A. health practitioner obtains a whole blood sample, a nasopharyngeal sample, and a blood sample in a Tempiss™ Blood RNA Tube (Applied Biosystems, Carlsbad, California) from the subject. A health practitioner orders a pathogen culture and a serum pathogen antigen detection test. A health practitioner orders at least one test to detemiiiie expression levels of the following endogenous genes: IFI27, ISG.15, OTOF, IF1T3, ITGAM, and ITGAX by .microarray hybridization assay. The pathogen culture is negative and the serum antigen test is positive for HHV-6. The microarray hybridization assay reveals a. decrease in expression of ITGAM, and ITGAX and an increase in expression of IFI27, ISGl 5, OTOF, and IF1T3. The patient is diagnosed with a symptomatic HHV-6 infection.
Example 16
This example illustrates a method of diagnosis of a viral pathogen-associated disease, A. patient exhibits symptoms that initially appear attributable to a viral or bacterial infection. A health practitioner obtains a. whole blood sample, a nasopharyngeal sample, and a blood sample in a Tenipus™ Blood RNA Tube ( Applied Biosystems, Carlsbad, California) from the subject. A health practitioner orders a pathogen culture, a serum pathogen antigen test, and serum antibody detection test. The health praciitioner additionally orders a test to determine expression levels of the following endogenous genes: ΜΎΗ9, ARAP3, CDK IC, T2A, SPATS2L 1RF7, ZB.P1 , OAS2, OAS ! , iS IS, IF16, H.ER.C5, OAS3, RSAD2, OAST, IF1T3, OTOF and IF127 by microarray hybridization assay. The pathogen culture is negative, the serum antibody test is negative, and the serum antigen test is positive for enterovirus. The roi.croa.rray hybridization assay reveals an increase in expression of
CD NIC MT2A. SPATS2L, IRF7, ZBF 1 ? OAS2, OASl , ISG15, W16, HB.RC5, AS3. RSAD2, OASL, !ΤΓΓ3, OTOF and IFI27 and a decrease in expression of MYH9 and ARAP3. The patient is diagnosed with a symptomatic enterovirus infection.
Example ! 7
Thi s example ill ustrates a method of diagnosi s of a pathogen-associated disease.
A patient exhibits symptoms that initially appear attributable to a viral or bacterial infection. A health practitioner obtains a blood sample in a Tempus™ Blood RNA. Tube (Applied Biosysteras, Carlsbad, California) from the subject The health practitioner orders a test to determine expression levels of the following endogenous genes: ΜΥΗ » ARAP3S CDKN IC, T2A, SPATS2L, IRF7, Z.BPI. OAS2, OAS l , ISG15. W16, HERC5, OA S3, RSAD2, OASL, IFIT3, OTOF and FI27 by niicroarray hybridization assay. The microarray hybridization assay reveals an increase in expression of CDKN IC, MT2A, SPATS2L, IRF7, ZBPT OAS2, OASl, ISG15, IFI6, H.ERC5, OAS3, RSAD2, OAS I,, IFIT3, OTOF and IFI27 and a decrease in expression of MYH9 and ARAP3. The patient is diagnosed with a symptomatic viral infection.
Example 1.8
This example illustrates a method of diagnosis of a pathogen-associated disease.
A patient exhibits symptoms that initially appear attributable to a viral or bacterial infection. A health practitioner obtains a blood sample in a Tempus™ Blood RNA "l ube (Applied Biosysteras, Carlsbad, California) from the subject. The health practitioner orders a test to determine expression levels of the following endogenous genes: MYH9, ARA.P3, CDKNIC, MT2A, SPATS2L, IRF7, ZBP1, OAS2, OASl , ISO 15, IFI6, HER.C5, OAS 3, RSAD2, OASL, IF1T3, OTOF and 1FI27 by niicroarray hybridization assay. The mic.roa.rray hybridization assay reveals a decrease in expression of CDKNIC. MT2A, SPATS2L, iRF7, ZBP.1, OAS2, OAS 1, ISO 15, IFJ6, HERGS, OA.S3, R.SAD2, OASL, IFIT3, OTOF and IFI27 and an increase in expression of MYH9 and ARAP3. The patient is diagnosed with a symptomatic bacterial infection.
Gene and transcript sequences are all known in. the art. Sequences are available from databases such as those of the Na.litraal Center for Biotechnology Information. All references cited are hereby incorporated by reference, each in its entirety..

Claims

What is claimed is:
1. A. method of diagnosis of a pathogen-based disease in a subject, comprising:
a) providing at least one biological sample from a human subject;
b) determining presence, absence and/or quantity of a bacterial pathogen, a viral pathogen, or a combination thereof, by a pathogen culture, a serum antibody detection test; a pathogen antigen detection test, a pathogen D A detection test, a pathogen RNA detection test, or a combination thereof;
c) determining in the at least one sample, expression levels of at least one endogenous gene in which aberrant expression levels are associated with infection with a pathogen, by a mieroarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay, an enzyme-linked immunosorbent assay (ELISA) or a combination thereof, whereby the subject is diagnosed with the disease if the subject has an aberrant level of expression of at least 1.5 fold increase or decrease of the at least one gene, wherein an aberrant level of expression of the at least one gene is associated with infection, with the pathogen.
2. A .method in accordance with claim 1 , wherein the at least one endogenous gene consists of one gene.
3. A method in accordance with claim ! , wherein the at least one endogenous gene consists of two genes.
4. A method in accordance with claim I , wherein the at least one endogenous gene consists of three genes.
5. A method in accordance with claim I , wherein the at least one endogenous gene consists of four genes.
6. A. method in accordance with claim Ϊ , wherein the at least one endogenous gene consists of five genes.
?. A method in accordance with claim 1 , wherein the at least one endogenous gene consists of six genes.
8. A method in accordance with claim I , wherein the at least one endogenous aene consists of seven genes.
9. A method in accordance with claim 1 , wherein the at least one endogenous gene consists of eight genes. 10, A method in accordance with claim 1, wherein the at least one endogenous gene consists of nine genes.
1 , A method in accordance with claim 1 , wherem the at least one endogenous gene consists of ten genes.
12, A method in accordance with claim 1, wherein the at least one endogenous gene consists of eleven genes,
13, A method in accordance with claim 1 , wherem the at least one endogenous gene consists of twelve genes,
14, A method in accordance with claim. 1 , wherei the at least one endogenous gene consists of thirteen genes.
15, A method in accordance with claim 1 , wherein the at least one endogenous gene consists of fourteen genes.
16, A method in accordance with claim 1 , wherein the at least one endogenous gene consists of fifteen genes,
17, A method in accordance with claim 1, wherein the at least one endogenous gene consists of sixteen genes.
18, A method in accordance with claim 1 , wherein the at least one endogenous gene consists of seventeen genes.
19, A method in accordance with claim 1, wherein the at least one endogenous gene consists of eighteen genes.
20, A method in accordance with claim 1 , wherein the at least one endogenous gene consists of nineteen genes.
21 , A method in accordance with claim. L wherein the at least one endogenous gene consists of twenty genes.
22, A method in accordance with claim 1 , wherein the at least one endogenous gene consists of twenty-one genes.
23, A method in accordance with claim 1, wherein the at least one endogenous gene consists of twenty-two genes.
24, A. method in accordance with claim 1 , wherein the at least one endogenous gene consists of twenty-three genes.
25, A method in accordance with claim 1 , wherein the at least one endogenous gene consists of twenty-four genes,
26, A. method in accordance wit claim 1, wherein the at least one endogenous gene consists of twenty-five genes.
27. A method in accordance with claim 1, wherein the at least one endogenous gene consists of twenty-six genes.
28. A method in accordance with claim 1, wherein the at least one endogenous gene consists of twenty-seven genes.
29. A method in accordance with claim 1, wherein the at least one endogenous gene consists of twenty-eight genes.
30. A method in accordance with, claim 1 , wherein the at least one endogenous gene consists of twenty- ine genes .
31. A method in accordance with claim 1, wherein the at least one endogenous gene consists of thirty genes.
32. A method in accordance wit claim 1 , wherein the at least one endogenous gene consists of thirty-one genes.
33. A method in accordance with claim 1 , wherein the at least one endogenous gene consists of thirty-two genes.
34. A method in accordance with claim 1, wherein the at least one endogenous gene consists of th irty-three genes.
35. A method in accordance with claim 1, wherein the disease is Fever Without an Apparent Source.
36. A method in accordance with claim 1, wherein the pathogen is a virus.
37. A method in accordance with claim 36, wherein the virus is selected from the group consisting of an adenovirus, aa enterovirus, a human herpesvirus 6 (EHV-6) and a rhinovirus,
38. A method in accordance -with claim. 1, wherein the pathogen is a bacterium.
39. A method in accordance with claim. 38, wherein the bacterium is selected from the group consisting of an Escherichia co!i, a Staphylococcus aureus, a Streptococcus pneumoniae and a combination thereof.
40. A method in accordance with claim 1, wherein the at least one endogenous gene is selected from the group consisting of IFI27, ISGi S, OTOF, IFH'3, ΠΧ3ΑΜ. and H'GAX,
41. A method in accordance wit claim. 1 , wherein the at least one gene is selected from the group consisting of OTOF and ITGAX.
42. A method in accordance with claim 1 , wherein the at least one gene is selected from the group consisting of IF 127 and ITGAM,
43. A method in accordance with claim 1 ? wherein the determining expression levels comprises a. real time polymerase chain reaction assay, a reverse transcriptase polymerase chain reaction assay, or a combination thereof
44. A method in accordance with claim lfwherein the determining expression levels comprises an oligonucleotide array assay, a probe hybridization assay a gene expression array assay, a cDNA microarray hybridization assay or a combination thereof.
45. A method in accordance with claim 1, wherein the microarray hybridization assay- consists essentially of .from I to 47,300 probes, or about 47,300 probes.
46. A method in accordance with claim 1 , wherein the microarray hybridization assay consists essentially of from I to 5700 probes, or about 5700 probes.
47. A method in accordance with claim 1, wherein the microarray hybridization assay consists essentially of from 1 to 50 probes, or about. 50 probes.
48. A method in accordance with claim 1, wherein the microarray hybridization assay consists essentially of from I to 10 probes, or about 10 probes.
49. A method in accordance with claim 1, wherein the microarray hybridization assay consists essentially of between 18 and 33 probes, or about 33 probes.
50. A method in accordance with claim 1, wherein the microarray hybridization assay consists essentially of from 1 to 260 probes, or about 260 probes,
51 . A method in accordance with claim 1 , wherein the microarray hybridization assay consists essentially of from I to 1321 probes, or about 132 probes.
52. A method in accordance with claim. 1, wherein the microarray hybridization, assay consists essentially of from 1 to 18. probes, or about 18 probes.
53. A method in accordance with claim 1 , wherein the microarray hybridization assay consists essentially of from I i 22 probes, or about 22 probes.
54. A method in accordance with claim 1, wherein the microarray hybridization assay consists essentially of from 1 to 33 probes, or about 33 probes.
55. A method in accordance with, claim 1, wherein the microarray hybridization assay consists essentially of from Ϊ to 260 viral response-specific probes or about 260 viral response-specific probes and from 1 io 1321 bacterial response-specific probes or about 1321 bacterial response-specific probes.
56. A method in accordance wit claim. 1 , wherein the microarray hybridization assay consists essentially of from I i 3467 HHV-6 response-specific probes or about 3467 HHV-6 response-specific probes.
57. A method in accordance with claim 1 , wherein the microarray hybridization assay consists essentially of from 1 to 464 HHV-6 response-specific probes or about 464 HHV-6 response-specific probes.
58. A method in accordance with claim 1, wherein the microarray hybridization assay consists essentially of from I to 798 HI iV-6 response-specific probes or about 798 ί il l V-6 response-specific probes.
59. A method in accordance with claim 1, wherei the microarray hybridization assay consists essentially of from I to 5604 adenovirus response-specific probes or about 5604 adenovirus response-specific probes,
60. A. method .in accordance with, claim 1 , wherein the microarray 'hybridization assay consists essentially of from I to 2078 adenovirus response-specific probe or about 2078 adeivo viro s response-specific probe .
61. A method in accordance with claim 1, wherein the microarray hybridization assay consists essentially of from I to 847 adenovirus response-specific probes or about 847 adenovirus r espoii se-speci.fi c probes.
62. A method in accordance with claim 1 , wherein the microarray hybridization assay consists essentially of from I to 4184 enterovirus response-specific probes or abou 4184 enterovirus response- pecific probes.
63. A method in accordance with claim 1 , wherein the microarray hybridization assay consists essentially of from I to 594 enterovirus response-specific probes or about 594 enterovirus response-specific probes.
64. A method in accordance with claim 1, wherein the microarray hybridization assay consists essentially of from 1 to 678 enterovirus response-specific probes or about 678 enterovirus response-specific probes.
65. A method in accordance -with claim. 1, wherei the microarray hybridizatio assay consists essentially of from 1 to 1234 bacterial, response-specific probes or about .1234 bacterial response-specific probes.
66. A method in accordance with claim 1 , wherein the microarray hybridization assay consists essentially of from I to 1939 bacterial response-specific probes or about 1939 bacterial, response-specific probes.
67. A method in accordance with claim 1 , wherein the at least one biological sample is selected from, the group consisting of peripheral blood mononuclear cells, a nasopharyngeal sample, a urine sample, a blood sample, a lumbar puncture sample, a bodily fluid, a biopsy sample, a tissue sample and a combination thereof.
68. A method in accordance with claim f wherei the at least one biological sample comprises a peripheral blood sample.
69. A method in accordance with claim 1, wherein the at least one biological sample comprises, consists essentially of, or consists of peripheral blood mononuclear cells.
70. A method in accordance with claim 1 , wherein the at least one endogenous gene is selected from the group consisting of Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses Pathway genes, TRBM I Signaling Pathway genes. Toll-like Receptor Signaling Paihway genes. Natural Killer Cell Signaiing Pathwa genes, Interferon Signaling Pathway genes. Activation, of interferon Regulatory Factors by Cytosolic Pattern. Recognition Receptors Pathway genes, Integrin Signaling Paihway genes and a combination thereof.
71. A method in accordance with claim. 1, wherein the at least one endogenous gene comprises, consists essentially of, or consists of an Interferon Signaling Pathway gene.
72. A method in accordance with claim 1 , wherein the at least one endogenous gene comprises, consists essentially of; or consists of a Integrin Signaling Pathway gene.
73. A method in accordance wi th claim 1 , wherein the human subject is a child.
74. A method in accordance with claim 73, wherein the human subject is between 0 to 36 months of age,
75. A method in accordance with claim 73, wherein the human subject is between 2 to 36 months of age.
76. A method of distinguishing a viral-caused, infection from a bacterial-caused infection or a combination thereof, comprising;
a) obtaining at least one biological sample from, a human subject;
b) determining presence, absence and/or quantity of a viral pathogen by a pathogen culture, a. serum antibody detection test, a pathogen antigen defection test, a pathogen DNA detection test, a pathogen RNA detection test or a combination thereof;
c) determining presence, absence and/or quantity of a bacterial pathogen by a pathogen culture, a serum antibody detection test, a pathogen antigen defection test, a pathogen DNA detection test, a pathogen RNA detection test or a combination thereof; and d) determining in the- at least one sample, ex pression level of at least one endogenous gene in which aberrant expression is associated with infection with a viral pathogen, and expression level of at least one endogenous gene in which aberrant expression is associated infection with a bacterial pathogen, by an assay selected from the group consisting of a microarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a
L AMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay, an enzyme-linked immunosorbent assay and a combination thereof, whereby the subject is diagnosed with a viral infection if the sample comprises a) a virus pathogenic for the infection and b) aberrant expression level of at least one endogenous gene associated with a viral infection, and whereby the subject is diagnosed with a bacterial infection if the sample comprises a) a bacterium pathogenic for the infection and b) aberrant expression level, of at. least one endogenous gene associated with a bacterial infection.
77. A method in accordance with claim 1. or claim 76, wherein the at least one gene associated with a bacterial infectio is selected from the group consisting o FYN, CD247, EIXPR3, CD3, ZAP70, PLCG L PR CH, IXK, LAX, PRK.CQ, ITK, REOti, GH 13, PPPI I2 , RHOTt FCER1G, LYN, RALB, GNAQ, MARCKS, XGM2, ARBGEFI L MYL12A, ΪΪΡ300, MYL9, GREB5, FCGR2A, GNG10, GNG.1 1 , C1QB, NOD2, TLR2, TLR I , RNASEL, C5AR1 , TLR4, MYD88, PIK3CB, C3AR1, T.LR6, CAS l, XLR5, NLRC4, TLR 8, IL1 B, ITGB7, TSPAM4, PPPI R ZYX, VASP, 1TGA2B, ITGB5, VCL ΓΓΟΒ3, MYLK, ASAP1 JTGAM, ITGAX. KLRDL IR2DL3, IR2DL4, KIR3DL3, KSR3DLL HCST, CD247, NCR3, FCGR3B, SIGLEC , FCER IG, JAK2, CA P5 and a combination thereof
78. A method in accordance with claim 1 or claim 76, wherein the at least one gene associated with a bacterial infection is selected from the group consisting of RBOU, GMA1.3, PPPIRI , RHOT1, FCE IG, LYN, RALB, Gl^ Q, MARCKS, TGM2, ARHGEF1 1 , MYL12A, EP300, YL-9, CREB5, FCGR2A, GN 10, GNOl 1 , C1QB, NOD2, TLR2, TLR I , RNASEL, C5AR1 , TLR4, MYD88, PIK3CB, C3AR1, TLR6, CASPL TLR5, NLRC4, TLR 8, if I B, PPPIRL ZYX, YLJ 2A, VASP. LTGA2B, ITGB5, VCL, ITGB3, MYLK, ASA L ITGAM, ITGAX, FCGR3B, SIGLEC9, FCE IG, JAK2, CASP5 and a combination thereof, wherein the at least one gene has an aberrant level of expression of at least a 1.5 fold increase compared to a control level.
79. A method in accordance wit claim 1 or claim 76, wherein the at least one gene associated with a bacterial infection is selected from the group consisting of FYN, CD247, EITPR3, CD3, ZA.P70, PLCGl, PR CH, LO , LAX, PRKCQ, ITK, ITGB7, TSPAN4, KLRDL KIR2DL3, K1R2DL4, KIR3DL3, KIR3DLL I ICS'L CD247, NCR3 and a combination thereof wherein the at least one gene has an aberrant level of expression of at least a. L5 fold decrease compared to a control level.
SO, A method in accordance with claim I or claim 76, wherein the at least one gene is a Amiexin A3 gene,
81. A method of diagnosis of a Fever Without an Apparent Source in a subject, comprising; a) providing at least one biological sample from a human subject; b) determining presence, absence and/or quantity of a bacterial pathogen, viral, pathogen, or a combination thereof, by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen ON A detection test, a pathogen RNA detection test, or a combination thereof;
c) determining in the sample, expression levels of at least one endogenous gene associated with aberrant expression levels resulting from infection with the pathogen, by a mieroarray hybridization assay, an R A-seq assay, a polymeras chain, reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot assay, an enzyme-linked immunosorbent assay or a combination thereof, whereby the subject is diagnosed with the disease if the sample comprises the pathogen and the sample exhibits an aberrant level of expression of at least one gene associated with aberrant expression levels resulting from infection with the pathogen.
S2, A method of diagnosis of a. pathogen-associated disease comprising:
a) providing at least one biological sample from a human subject;
b) determining presence, absence and/or quantity of a bacterial pathogen, viral pathogen, or a combination thereof by an assay selected from the group consisting of a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen DNA detection test, & pathogen RNA detection test and a combination, thereof; c) determining in the sample, expression levels of at leas one endogenous gene selected from the group consisting of IP127, ISG15, OTOF, IFIT3, 1TGAM, 1TGAX and a combination thereof, by an assay selected from the group consisting of a mieroarray hybridization assay, an. RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blot- assay, an enzyme-linked immunosorbent assay, or a. combination thereof, whereby the subject is diagnosed with the disease if the sample comprises the pathogen, and the sample exhibits an aberrant level of expression of at least one gene selected from the group consisting of 1FI27, ISO 15, OTOF, ΙΡΓΓ3, ITGA , JTGAX and a combination thereof
83, A method of diagnosis of a viral pathogen-associated disease comprising:
a) providing at least one biological sample from a human subject;
b) determining presence, absence and/or quantity of a vims selected from the group consisting of an adenovirus, an enterovirus, HH V-6, or a combination thereof by a pathogen culture, a serum antibody detection test, a pathogen antigen detection test, a pathogen D A detection test, a pathogen RNA detection test, or a combination thereof; c) determining in the sample, expression level of at least one endogenous gene that exhibits aberrant expression during infection with the vims, by an assay selected from the gro up consisting of a mieroarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a
Northern blot assay, a Western blot assay, an enzyme- linked immunosorbent assay and a combination thereof, whereby the subject is diagnosed with the disease if the sample comprises a) the virus and b) an aberrant expression level of the at least one gene.
84. A method of diagnosis of a pathogen-associated disease comprising:
a) obtaining at least one biological sample from a human subject; and
b) determining, .in the sample, expression levels of at least one endogenous gene selected from the group consisting of IF 127, IS015, OTOF, IF1T3, IT AM, ITGAX and a combination thereof, by a mieroarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a. LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western blo assay, an enzyme-linked immunosorbent assay or a combination thereof, whereby the subject is diagnosed with the disease if the sample exhibits an aberrant level of expression of at least one gene selected from, the group consisting of IF.S27, ISO 15, OTOF, IFIT3, ITGAM, ITGAX, and a combinatio thereof
85. A method in accordance with claim. 84, wherein the disease is Fever Without an. Apparent Source,
86. A method in accordance with claim 84, wherein the biological sample comprises peripheral blood mononuclear cells,
87. A method in accordance with claim. 1, wherein the at least one aberrant level of expression is at least a 2 fold increase or decrease compared to a control level.
88. A method in accordance with claim 1, wherein the at least one aberrant level of expression i at least a 1.5 fold increase or decrease compared to a control level.
89. A method in accordance with claim 1, wherein the at least one gene associated with a adenoviral Infection is selec ted from the group consisting of ATM, PEKQl, PRKCQ, C 1QB, C1QC, IRF7, OAS3, OAS I , OAS2, A 2, EIF2, IFIHl, DDX58, NOD2, TIE 5, NLRC4, TLR8, C3AR1 , ILi B, TL 1 , TLR4, TLR6, MYD88, CASP! , 1F1T3, IFI35, BCL2, MED 14, IF GR2, IFNAR 1 , IRF1, 8 AX, IRF9, FSMB8, IFITMI, JA 2, STAT2, TAPl, IKBKB. IKBKAP, IAA127 L TRAF6, I NF, TB 1, TANK, IRF9, NFKBIA, 1RF7, 1SG 15, ADAR, ZBP1 , IF.IT2, .FOS, LY96, TLR5, IRA.K3, TLR8., E1F2AK2 CD 14, MARK 14, STAT4, HS.572649, CCR7, CD4GLG, LTB, HLA-DOA, CREB5, FCCIR3B, FCGR2A, IL1 RN, LTBR, TYROBP, FCE 1G, FCGR A, PCGR IB and a combination thereof.
90. A method in accordance with claim 1, wherein the at least one gene associated with an adenoviral infection is selected from the group consisting of C 3 QB, CiQC, IRF7, OA.S3, OAS E OAS2, ΛΚ2, B3F2, IFIH 1, DDX58, NOD2, TLR5, NERC4, TER8, C3AR I , ilJB, TLR1 , TLR4, TLR6, MYDSS, CASP1 , ΙΡΓΒ, ΪΡΙ35, BCI.2, IF AR1 , IRF.l , BAX, IRF9, PSMB8, 3FFFM 1 , JAK2, STAT2, Ί API , TRAF6, TNF, I'BKJ , TANK, JRF9, NF BIA, IRF7, ISG15, A AR, ZBPE IFIT2, FOS, LY96, TLR5, IRAK3, TLR8, EIF2AK2, CD 14, MAPK.14, CREB5. FCGR3B, FCGR2A, 1L1R , LI BR, TYROBP, FCER.1G, FCGR1 A, FCG 1B and a combination thereof, wherein the at least one gene has an aberrant level of expression of at least a 1.5 fold increase compared to a control level.
91. A method in accordance with claim 1, wherein the at least one gene associated with an adenoviral infection is selected from the group consisting of ATM, PRKCFL PRKCQ,
ED .14, IFNGR2, IKBKB, !KBKAP, K1AA127J , STAR, HS.572649, CCR7, CD40LG, ETB, HLA-DOA and a combinatio thereo wherein the at least one gene has a aberrant level of expression of at least a 1.5 fold decrease compared to a control level
92. A method in accordance with claim 1, wherein the at least one gene associated with a entero viral infection is selected from the group consisting of NLEC4, TER8, TI..R5, NOD2, RNASBL, TLR2, TLR'I , C5AR 1, MYD88, TLR6, CAS.P1» SUB, C! QB, IRF7, OAS ! , OAS3, E1F2AK2, DDX58, ΠΤΗ3 , OAS2, 1E3T1, IF D, IF135, MXl, TAP E STAT2, IFiT l, ST ATI , JA 2, IRF7, ISG i S, IFIT2, DHX58, ZBPl, FOS, LY96, MAP2R3, MAPK.14, CASP5 and a combination thereof.
93. A method in accordance with claim 1, wherein the at least one gene associated with an enierovirai infection is selected from the group consisting of 1..RC4, TLRS, TLR5. NO.D2, RNA.SEL, TLR2, TLR.E C5AR3 , MYDSS, TER6, CASPE IL1 B, C1QB, IRF7, O.AS1, OAS3, EIF2A 2, DDX58, IFIHl , OAS2, IFLTE IF1T3, IFB5, MX I , TAP!, STAT2, IFFTM1 , STATE JAK2, 1RF7, ISGI 5, ΪΕΙΤ2, DHX58, ZBPl , FOS. LY96, MAP2K.3, MAPKI4, CASP5 and a combination thereof, wherein the at least one gene has a aberrant level of expression of at least a 1.5 fold increase compared to a control level.
94. A method in accordance with claim. 1, wherein the at least one gene associated with an HHV-6 infection is selected from the group consisting ofCCLS, PR CH, TLRS, TLR2, NOD2, TLRS, TLR4, TLR6, C1QA, MYDSS, R1PK2, IL B, C3AR3 , CASPl, CTQB, CIQC, OASi, IRF7, OAS3, E1F2A 2, TLR7, OAS2, DDX58, FPll i i . IFFB, IFOT , SOCS l s MXl, 1FI35, IFiTM l , TAP E STAT2, JAK2, STAT 3 , TYROBP, CD86, ΙΠ Β, CASP5, CCL2, TLR.9, TRAF6, I A 4, CHUK, CD 14, MYDSS, TLR1 , FOS, L.Y96 and a combination thereof. 95, A method in accordance with claim 1, wherein the at least one gene associated with an HHV-6 iniection is selected from the group consisting of TLR5, TLR2, NOP2, TLR8, TLR4, TLR6, CIQA, YD88, RIPK2, ILiB. C3AR1. CASPl , C 1QB, C IQC, OAS L iRF7, OAS3, EIF2AK2, TLR7, OAS2, DDX58, IFIHl , IFIT3, IFIT1, SOCS1, MX 1. 1FI35, IF1T I, TAP 1, STAT2, JA 2, STAI R TYROBP, CDS6, ILIB, CASP5, CCL2, TRAF6, 1RAK4, CBUR, CD 14. MYD88, TLRL FOS, LY96 and a corobination thereof, wherein the at least one gene has an. aberrant level of expression of at least a .1 ,5 fold increase compared to a control level
96, A method in accordance with claim. 1, wherein the at least one gene associated with an HHV-6 infection is selected from the group consisting of CCL5, PRKCH and TLR9, wherein the at. l east one gene has an aberrant. level of expression of at least a 1.5 fold decrease compared to a control level,
97, A method in accordance wi th claim 1 or claim 76, wherein the at least one gene is selected from the group consisting of ΜΥΉ9, ARAP3, C KNtC, MT2A, SPATS2L, 1RF7, ZBPL OAS2, OAS L ISGI5, IP16, HRRC5, OAS3, RSAD2, OASL, IFIT3, OTOF and IFT27.
98, A method in accordance with claim 1 or claim 76, wherein the at least one gene associated with a viral infection is selected front the group consisting of CD N1C, MT2A, SPATS2L, IRF7, ZBPL OAS2, OAS1 , 1SG15, F16, HERC5, OAS3, RSAD2, OASL, IFIT3, OTOF and ΙΡΓ 7, wherein the at least one gene has an aberrant level of expression of at least a 1.5 fold increase compared t a bacterium-infected control level,
99, A method in accordance with claim 1 or claim 76, wherein the at least one gene is selected from the group consisting ofMYH9 and ARA.P3, wherein, the at least one gene has an aberrant level of expression of at least a 1.5 fold decrease compared to a bacterium- infected control level.
100, A method in accordance with claim 1 or claim 76, wherein the at least one gene is selected from the group consisting of B AR L RRAS, ACTR2, NCK2, PIK3CB, MAP2 4, 1TGA2B, ITGB3, MYLK, YL , ITGB5, GNGI L ZYX, I GAX, IF GR1 , ITGAM, ST.AT2, IFI35, MX i , OAS I , IFIT1 and IFIT3.
101 , A method in accordance with claim 1 or claim 76, wherein the at least one gene associated with a viral infection is selected from the group consisting of BAKL 1F G L STAT2, 1F135, MXl , OAS1, l! and IFIT3, wherein the at least one gene has an aberrant level of expression of at least a 1.5 fold increase compared to a bacterium-infected control level.
102. A method in accordance with e!aini 1 or claim 76, wherein the at least one gene is selected .from the group consisting of RRAS, ACT 2, NC 2, FI 3CB, MA P2K.4, ITGA2B, ITGB3., MYL , MYL9, ITGB5, GNGI 1 , ZYX, ITGAX and ITGAM, wherein the at least one gene has an aberrant level of expressio of at least a 1.5 fold decrease compared to a bacteri um -in fected control, level .
103. A method in accordance with claim 1 or claim 76, wherein the at least, one gene is selected from the group consisting of OSBPL8, VHL, ACTR2, MAP2K4, F13A1, PROS 1, 1TGB3, MYL9, ITGA2B, ITGB5, GNG1 1, EP3O0, ZYX, ARAP3, AGER, ITGAX, SORLL IFNGRI , IFNGR2, ITGAM, MT2A, SPATS.2L OAS2, OA l , ISO 1.5, IF 6, IF TJ , MERCS, GAS3, RSAD2, OASL, OTOF and IF127.
.1 4, A method in accordance with claim 1 or claim 76, wherein the at least one gene is selected from, the group consisting of IFNGRI , IFNGR2, MT2A, SPATS2L, OAS2, OASl, ISG15, IFI6, !FITI , HERC5, OAS3, RSAD2, OASL, OTOF antl 1FI27, wherein the at least one gene has an aberrant level of expression of at least a 1.5 told increase compared to a bacterium-infected control level
105. A method in accordance with claim I or claim 76, wherein the at least one gene is selected from the group consisting of OSBPL8, VHL, ACTR2, AP2 4, F13A 1 , PROS L If GEO, MYL9, ITGA2B, 1TGB5, GNGI 1 , EP300, ZYX, ARAP3, AGER, ITGAX, SORLI and IGTAM, wherein the at least one gene has an aberrant level of expression of at least a 1 ,5 fold decrease compared to a hacteri um~inf ected control level.
1 6. A method in accordance with claim 1 , wherein the at least one gene is selected from the group consisting of IFI27, ISG15, OTOF and 1FIT3, whereb the subject is diagnosed with a viral infection if the subject has an aberrant levei of expression of at least a 1.5 fold increase of at least one gene selected from the group consisting oiTFI27, ISG15, OTOF and IFIT3, compared to bacterial-infected control level
107. A method in accordance wit claim 1 , wherei the at least one gene is selected from the group consisting of IFS27, IF1T3, and a combination thereof, whereby the subject is diagnosed with febril e HHV-6 if the subj ect has an aberrant level of expression of at least a 1.5 i id increase of the at least one gene selected from the group consisting of IF 127 and 1FIT3, compared to an afebrile HHV-6 control level or a febrile control level.
108. A method in accordance with claim 1, wherein, the at least one gene is selected from the group consisting of IF1.27, ISO 1.5, and a combinatio thereof, whereby the subject is diagnosed with febrile adenovirus if the subject has an aberrant level of expression of at least a 1.5 fold increase of the at least one gene selected from the group consisting of IF127 and iSG! 5, compared to an afebrile adenovirus control level or a febrile control level,
09. A method in accordance with claim , wherein the at least one gene is selected from the group consisting of IF 127, ISG1.5 and IFIT3, whereby the subject is diagnosed with febrile enterovirus if the subject has an aberrant level of expression of at least a 1..5 fold increase of the a least, one gene selected from the group consisting of IFI27, ISG15 and IFIT3, compared to an afebrile enterovirus control leve! or a febrile control level,
3 10, A method in accordance with claim 1 or 76, wherein the at least one gene Is selected from the group consisting of ITGAM and ITGAX, whereby the subject is diagnosed with a bacterial infection if the subject has an aberrant level of expression of at least a 1.5 fold increase of the at least one gene selected from the group consisting of ITGAM and FTGAX, compared to an afebrile control level or a viral-infected control level,
1 1 1. A method in accordance with claim L wherein diagnostic accuracy is at. least 70%,
1 12, A method in accordance with claim L wherein diagnostic accuracy is at least 75%.
1 13, A method in accordance with claim I , wherein diagnostic accuracy is at least 80%.
1 1 . A method in accordance with claim 1 , wherein diagnostic accuracy is at least 85%. 1 15, A method, in accordance with claim 1 , wherein diagnostic accuracy is at least 90%. 116. A method of determining etiology of Fever Without an Apparent Source (FWS) In a subject, comprising:
a) providing a peripheral blood sample from a hum n subject;
b) determining presence, absence and/or quantity of a bacterial pathogen, a viral pathogen, or a combination thereof by a pathogen culture, a serum antibod detection test, a pathogen antigen detection test, a pathogen DNA detection test, a pathogen RMA detection, test, or a combination thereof;
c) determining in the sample, expression levels of at least one endogenous gene i which aberrant expression levels are associated with infection with a pathogen, by a microarra hybridization, assay, an RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a iigase chain reaction assay, a Southern blot assay, a Northern blot assay, a Western, blot assay, an enzytne-liiiked immunosorbent assay (BLISA) or a combination thereof, wherein the etiology of the disease is determined if the subject comprises the pathogen and the subject has an aberrant level of expression of at least 1.5 fold Increase or decrease of at least one gene, wherein an aberrant level of expression of the at least one gene is associated with infectio with the pathogen.
117. An antibiotic for use in a method of treatment of Fever without an Apparent Source (FWS) hi a subject, characterized in that the subject has in a peripheral blood sample at least a i .S fold increase in expressio level compared to a control level of at least one endogenous gene selected Irom the group consisting of FYN . CD247, E1XPR3, CD3, ZAP70, PLCG L PRKGH, LCK, LAI', PRKCQ, IT , RHOU, G A13, PPP1 R12A, RHOT1 , FCER1 G, LYN, RALB, G AQ, MARCKS, TGM2, ARHGEFI L MYL12A, EP300, MYL9, CREB5, FCGR2A, GNGIO, GNGU, CiQB. NODS', TLR2, TLRl, RNASEL, C5AR1 , TLR4, MY 88, PIK3CB, C3AR1, T.LR6, CASPl, TLR5, NLRC4, TLR8, ILIB, 1TGB7, TSPAN4, PPP! RI , ZYX, VAS.P, ITGA2B, JTGB5, VCL, ITGB3, MYLK, ASAP1. ITGAM, ITGAX, KL.RD1, KIR2DL3, KIR2DL4, KIR3DL3, 1R3DL1 , HCST, CD247, NCR3, FCGR3B, SIGLEC9, FCER'I , JAK2, CASP5 and a combination thereof.
1 1 S. An antibiotic for use in a method of treatment of Fever without an Apparent Source (FWS) in a subject, characterized in that the subject has hi a blood sample at least a 1.5 fold increase in expression level compared to a control level of a least one endogenous gene selected from the group consisting of RHOU, GNA 13, PPP1 12 , RHOT1. FCERIG, LYN, RALB, GNAQ, MARCKS, TGM2, ARHGEFH, MYLI2A, EP300, MYL9, CREB5, FCGR2A, GNG10, G G I L C I QB, NOD2, TLR2, T.L L RNASEL, CSA 1 , TLR4, MYD88, PIK3CB, C3AR.I , TLR6, CASPl , TLR5, NLR.C4, TLR8, ILI B, PPPI L ZYX, MYL12A, VASP, 1TGA2B, ITGB5, VCL, LTGB3, YLK, ASAP i, 1T AM, ITGAX, FCGR3B, SIGLEC9, FCE IG, JAK2. CASP5 and a combination thereof
1 1 . An antibiotic for use in a method of treatment of Fever without an Apparent Source (FWS) in a subject, characterized in that the subject has in a blood sample at least a 1.5 ibid increase in expression level compared to a control level of at least one endogenous gene selected from the group consisting of FYN, CD247, EITPR3, CD3, ZA 70, PLCG l , PRKCH, LCK, LAX, PRKCQ, ΓΓΚ, ITGB7, TSPAN4, LRD.1 , KJR2DL3, IR2DL4,
IR3DL3, IR3DL1, HCST, CD247, NCR3 and a combination thereof
120. An antibiotic for use in a method of treatment of Fever without an Apparent Source (FWS) in a subject, characterized in that the subject has in a blood sample at least a 1.5 fold decrease in expression level compared to a control level of Aimexm A3 gene.
121. An anti-viral drug for use in a .method of treatment of Fe ver with an Apparent Source (FWS) in a subject, characterized in that the subject has in a blood sample at least a 1.5 fold increase in expression level compared to a control level of at least one endogenous gene selected from the group consisting of IFI27, IS 15, OTOE, IFIT3, ITGAM, ITGAX and a combination thereof,
122. An ants -viral drug for use in a method of treatment of an adenoviral Infection in a subject, characterized in that the subject has in a blood sample an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of ATM, PRKCH, PRKCQ, CIQB, C1QC, IRF7, OAS3, OASL OAS2, AK2, ESF2, iFIH l, D.X58, NOD2, TLR5, NLRC4, TLR8, C3AR 1,
II I B, TLRI, TLR4, TLR6, MYD88, CASPl , IF1T3, 11 1. 5. BCL2, MED 14, IFNGR2, IFNAR L IRFL BAX, IRF9, PSMB8, IFITML JAK2S 5TAT2, TAP 1 , 1KBKB, ΪΚΒΚΑΡ, KIAA1271 , TRAF6, TNF, TBKL TANK, JRF , NPKBIA, JRF7, ISG 15, ADAR, ZBPL 1FS 2, FOS, LY96, TLR5, !RA . TLR.8, EIF2AK2, CD I 4, MA.PK.14, STAT4, HS.572649, CCR7, CD40LG, LI B, HLA-DOA, CREB5, FCGR3B, FCGR2A, ILIRN, LTBR, TYROBP, FCERIG, PCGR1A, FCG l B and a combination thereof.
123. An anti-viral drug for use in a method of treatment of an adenoviral infection In a subject, characterized hi that the subject has in a blood sample an aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of C 1 QB, CiQC, 1RF7, OAS3, OAS1 , OAS2, AK.2, EIF2, 1FIH I, DDX5S, NOD2, TLRS, NLRC , TLRS, C3AR L II. IB, TLR1 , TLR4, TLR6, MYD88, CASPL IF1T3, 1FI35, BCL2, IFNARL IRFi, BAX, IRF9, PSMB8, IFlTM i , JA 2, STAT2, TAP! , TRAF6, TNF, TBK1 , TANK, 1RF9, NFKB1A, IRF7, ISGl 5, ADAR, ZBPL IFIT2, FOS, LY96, TLRS, SRAK3, TLR8, EIF2A 2, CD 14, AP 1 , CREB5, FCGR3B, FCGR2A, ILIRN, LTBR. TYROBP, FCBRI G, FCG l A, FCGRIB and a combination thereof.
1.24. An anti-viral, drug for use in a method of treatment of an adenoviral infection in a subject, characterized in that the subject has in a blood sample an aberrant level of expression at least a L5 fold, greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of ATM, PRK.CFL PRK.CQ, MED14, EFNGR2,
I BKB, IKBKAP, IA 1271, STAT4, HS.572649, CCR7, CD40LG, LTB, HLA-DOA and a combination thereof and a combination thereof
1.25. An anti-viral drag for use in a method of treatment of an enteroviral infection in a subject, characterized in that the subject has in a blood sample an aberrant level of expression, at least a 1.5 fold greater compared to a control level of at least one endogenous gene selected from the group consisting of NLRC4, TLRS, TLRS, NOD2, R ASEL, TLR2, TL
C5AR 1, MYD88, TLR6, CAS l , IL LB, C1QR, IRF7, OAS I, OAS3, EIF2AK2, DDX58, IFIHl , OAS2, IFITL IFIT3, IFL35, MX L TAPL STAT2* IFIT L ST ATI, JAK2, IRF7, ISO 15, MT2, DMX58, ZBP1 , FOS, LY96, MAP2K3, MAPK14, CASP5 and a combination thereof.
126. An anti-viral drug for use in a method of treatment of a enteroviral infection in a subject characterized in that the siibjeci has in a blood sample a aberrant level of expression at least a 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of CCL5, PRKCH, TLR5, TLR2, NOD2, TL 8, TLR.4, TLR.6, C.1.QA, MY 88, R.1PK2, [LIB, C3AR.1 , CASP1 , C1QB, CIQC, AS 1, IRF7, OAS3, E1F2AK2, TLR7, OAS2, DDX58, IF!HL 1FFF3, IFTTL SOCSi, X1, IFB5, 1ΡΊΤΜ1, TAPL STAT2, JAK2, S AT.1 , TYR.O P. CDS6, IL1 B, CASP5, CCI.2, TLR9, TRAF6, IRAK4, CFRJK, CD 14, MYD88, TLR1, FOS, LY96 and a combination thereof.
127. An anli-viral drug for use in a method of Ireatment of an HHV-6 infection in a subject, characterized in that the subject has in a blood sampl an aberrant level, of expression at least a 1.5 fold grea ter or lesser compared to a control level of at least one endogenous gene selected from the gr up consisting of CCL5, PRKCH, TLR5, TLR2-, NOD2, TLR8, TLR4, TLR6, CI OA, MYD88, RIPK2, IL1B, C3AR1 , CASP1 , C1QB, C1QC, OAS 1, IRF7, OAS3, EIF2AK2, TLR7, OA.S2, DDX58, IFIH L lFiI'3, 1FIT1, SOCSI , MXl , IFB5, 1Ι ΓΜ1 , TAP1 , STAT2, JA 2, STAT'l , TYROBP, CD86, !LI B, CASP5, CCL2, T.LR9, TRAF6, 1RAK4, CHUK, CD14, MYD88, TLR'l, FOS, LY'96 and a combination thereof.
128. An anti-viral drug for use in a metliod of ireatment of an HFfV-6 infection in a subject, characterized in that the subject has in a blood sample an aberrant level of expression at least a 1.5 fold greater compared to a control level of at least one endogenous gene selected from the group consisting of TI ES, TL.R2, NOD2, T.LR8, TLR4, TLR6, CIQA, MYD88, RIPK2, IL1.B, C3AR L CASPL CIQB, CI QC, OAS I, IRF7, OAS3, EIF2AK2, TLR7, OAS2, DDX58. IFiH l , IFiT35 JFITl, SOCSI , MXL IFI35, IFFTM I , TAPl, STAT2, JAK2, STAT1, TYROBP, CD86, ILI B, CA.SP5, CCL2, TRAF6, 1RAK4, CF1UK, CD14, MYD88, TLR.l , FOS, LY96 and a combination thereof.
1.29. An anti-viral drug for use in a method of treatment of an HHV-6 infection in a siibjeci, characterized in that the subject ha in a blood sample an aberrant level of expression at least a 1 ,5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of CCL5, PRKCH, TLR9 and a combination thereof. 130. An anti-viral drug for use in a method of treatment of an HHV-6 infection in a subject, characterized in thai the subject has in a blood sample a aberrant level of expression at least 1.5 fold increase compared to a control level of at least one endogenous gene selected from the group consisting of CDKN1 C, MT2 , SPATS2L, FRF?, ZBPl , OAS2s OAS I, ISO 15, IF16, HERC5, AS3, RSAD2, OAS I . !FTD, OTOF and IFi27.
131. An anti-viral drug for use in a method of treatment of an HHV-6 infection in a subject, characterized in that the subject has in a blood sample an aberrant level of expression at least 1.5 fold decrease compared to a control le vel of at least one endogenous gene selected from the group consisting of MYH9 and ARAP3,
132. An anti-viral drug for use in a method of treatment of an HHV-6 infection in a subject, characterized in that the subject has i a blood sample an aberrant level of expression at least 1.5 fold increase compared to a control level of at least one endogenous gene selected from the group consisting of CD N1 C, MT2A, SPATS2L, 1RF7, ZBPl , OAS2, OAS F, ISO 15, IFI6, HERC5, OAS3, RSAD2, OASL, IFIT3, OTOF and IFI27 and at least 1.5 fold decrease compared to a control level of at least one endogenous gene selected from the group consisting of YH9 and ARAP3.
133. An antibiotic for use in a method of treatment of a bacterial infection in a subject., characterized in that a blood sample of the subject has at least 1.5 fold decrease in expressio of at least one endogenous gene selected from the group consisting of CDKNIG, MT2A, SPATS2L, IRF7, ZBPl , OAS2, OAS1, ISO 15, 1F16, HERC5, OAS3, RSAD2, OASL, IFIT3, OTOF and 1F127.
134. An antibiotic for use in a method of treatment of a bacterial infection in a subject, characterized in. that a blood sample of the subject has at least 1.5 fold increase in expression of at least one endogenous gene selected from the group consisting of MYH9 and ARAP3. 1.35. An antibiotic for use in a method of treatment of a bacterial infection in. a subject, characterized in that a blood sample of the subject has at least 1.5 fold decrease in expression of at least one endogenous gene selected front the group consisting of CDKNIC, MT2A, SPATS2L, 1RF7, ZBPl , OAS.2, OASL ISGI5, IF 16, HER.C5, OAS3, .SAD2, OASF, IF1T3, OTOF and IF127, and at least 1,5 fold increase in expression of at least one endogenous gene selected from the group consisting of MYB9, ARAP3 and a combination thereof.
136. An antibiot ic for use in a method of treatment of a bacterial infection, characterized in that a. blood sample of the subject has aberrant level of expression at least 1 ,5 fold greater or lesser compared to a control level of at least one endogenous gene selected from the group consisting of FYN, CD247, EITPR3, CD3, ZAP70, PLCGl, PR CH, LO FAT. PRKCQ, 1TK, RHOU, GNA J.3, PPP 1 R 12A, RHOT1, FCER1 G, LYN, RALB, GNAQ, MARC S, TGM2, ARHGEFI 1 , MYL12A, EP300, YL9, CREB5, FCGR2A, GNG10, GMG1 1 , C1 Q.B, NGD2, TLR2, TLR1, RNASEL, C5ARI , TLR4, MYDSS, PI CB, C3AR1 , TLR6, CASPl , TLRS, NLRC4, TLRS, IL1 B, ITGB7, TS.PAN4, PPP1 R1 , ZYX, VAS.P, ETGA2B, Γ ΟΒ5, VCL, ITG B3, MYLK, ASAPL Π GAM, 3TGAX, LRDL IR2DL3, IR2DL4, KIR3DL3, K1R3DLI, HCST, CD247, CR3, FCGR3B, S1GLEC9, FCER 10, JAK2, CASH and a combination thereof
137. An. antibiotic for use in a method of treatment of a bacteria! infection, characterized in thai the subject has aberrant level of expressio of at least one endogenous gene selected from the group consisting of RMOU, G!SiAJ '3, PPP1 R12A, REOT3, FCERIG, LYN, RALB, GNAQ, MARCKS, TGM2, ARHGEFI l , MYL12A, ΕΡ3Θ0, MYL9, CREB5, FCGR2A, GNG10, GNG1 .U C1.QB, NOD.2, TLR2, TLR1, RNASEL, C5AR1, TLR4, MYD8S, PiJ CB, C3AR1 , TLR6, CASPl , TLRS, N.LRC4, TLRS, ILl B, PPPI R I , ZYX, MYLI2A, VASP, ITGA2B, ITGB5, VCL 1TGB3, MYLK, ASAPi , ITGA , !TGAX, FCGR3B, S1GLBC9, FCERIG, JAK2, CASP5 and a combination thereof
1 8, An anti-viral drug for use in a method, of treatment of an enteroviral infection in a subject, characterized in that the subject has in a blood sample an aberrant level of expression at least 1.5 fold greater or lesser compared to a control level of at least one endogenous gene selected from, the group consisting of adenoviral infection is selected from the group consisting of NLRC4, TLRS, TLRS, NOD2, RNASEL, T.LR2, TL L C5AR1 , MYD88, TLR6, CASPl , 1L1 B, C1 QB, LRF7, OAS1 , OAS3, E1F2AK2, DD 58, ΙίΤίΟ , OAS2, ΪΙ Π, If ΪΤ3, IFI35, MXl, TAP 1 , STAT2, IFITM i , STAT'i , JA 2, IRF7, IS01 5, IF1T2, D 11X58, ZBP L FOS, LY 6. MAP2 3, MAPK1.4, CASP5 and a combination thereof
139, An anti-viral drug for use in a method of treatment of an enteroviral infection in a subject characterized in that the subject 'has in a blood, sample an aberrant level of expression at least 1.5 fold increase compared to a control level, of at least one endogenous gene selected from the group consisting of NLRC4, TLR8, TLRS, NOD2, RNASEL, TLR2, TLR L C5ARL MYD88, TLR6, CASPL ILI B, CIQB, IRF7, QASL OAS3, E1F2A 2, DDX58, IPIHI , OAS2. lElTL IFIT3, 1FI35, MXl, TAP!, STAT2, IFITMI, STAT1, JAK2, IRE7, ISO 15, DFIT2, DHX58, ZBP1, FOS, LY96, MAP2 3. MAPK34, CASP5 and a combination thereof,
140, An anii-vira! drug for use in a method of treatment of an BHV~6 infection in a subjects characterized in that the subject has in a blood sample an aberrant level of expression at least 1 ,5 fold decrease compared to a control level of at least one endogenous gene selected from the group consisting of CC.L5, PRKCFS and TLR.9.
141 , An anii-vira! drug for use in a method of treatment of a viral infection in a subject, characterized in that the subject has in a blood sample an aberrant level, of expression at least
1.5 fold increase of at least one endogenous gene selected from the group consisting of CDK 1C, MT2A, SPATS2L, 1RF7, ZBPU OAS2, OAS J , ISO 15, 1FI6, HERC5, OAS3, SAD2, OASL, IFFB, OTOF and IFI27, compared to a bacterium-infected control level. 1.42. An anti-viral drug for use in a method of ireatment of a viral infection in a subject, characterized in that the subject has in a blood sample an aberrant level of expression at least 1.5 ibid decrease of at least one endogenous gene selected irom the group consisting of YH9 and ARA.P3, compared to a haeteriuni-infecied control level.
143. An anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in that the subject has in a blood sample an aberrant, level of expression at least a 1.5 fold increase of at least one endogenous gene selected from the group consisting of BAK1, IFNGR1, STAT2, IFI35, Xf OAS1, IFiTi and lFiT3, compared to a bacterium- infected control level.
144. An anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in that die subject has in a blood sample an aberrant level of expression at least a 1.5 ibid decrease of at least one endogenous gene selected from the group consisting of RRAS, ACTR2, NCK2, PIK3CB, MAP2K4, ITGA2B, ITGB3, MYLK, MYL9, 1TGB5, GN 11, ZYX, ITGAX and ITGAM, compared to a bacterium-iniected control level
145. An anti-viral drug for use in a method of ireatment of a viral infection in a subject, characterized in that the subject has in a blood sample an aberrant level of expression at least a 1.5 fold increase of at least one endogenous gene selected from the group consisting of 1FNGRL 1FNGR2, MT2A, SPATS2L, OAS2, OAS1 , 1SG15, ΙΡΊ6, ΙΡΤΠ , HERC5, O.AS3. RSAD2, OASL, OTOF and. IPt27, compared to a bacterium-infected control level,
146. An anti-viral drug for use in a method of treatment of a viral infection in a subject, characterized in that the subject ha in a blood sampl e an aberrant level of expression at least a 1 -5 fold decrease of at least one endogenous gene selected from the group consisti ng of OSBPL8, VHL, ACTR2, MAP2 4, F13AL PROSI , ITG.B3, MYL9, ITGA2B, JTGB5, GNG.1.1 , EP300, ZYX, ARAP3, AGER, ITGAX. SORT I and IGTAM, compared to a bacterium-infected control level.
147. An anti-virai drug tor use in a method of treatment' of a viral infection in a subject, characterized, in that the subject has in a blood sample an aberrant level of expression at least a 1.5 fold increase of at least one endogenous gene selected from the group consisting of 1F127, ISO 15, OTOF and 1FFD, compared to a bacterium-infected .control level
148. An anti-viral drug for use in a method of treatment of febrile HHV-6 in a subject, characterized in that the subject has been selected to have in a blood sample at least a 1.5 fold increase of at least one endogenous gene selected from the group consisting of IFJ27, IF1T3, and a combination thereof compared to an afebrile HHV-6 control level or a febrile control level.
1.49, An anti-viral drug for use in a method, of treatment, of febrile BHV-6 in a subject, characterized in that the subject has been, selected to have in a blood sample at least a 1.5 fold increase of at least one endogenous gene seleeied from the group consisting of IFI27, ISG i 5. and a combination thereof compared to an afebrile adenovirus control level, or a febrile control level.
150. An anti-viral, drug for use in a method of treatment of febrile enterovirus in a subject, characterized in that the subject has been, selected to have in a blood sample at least a 1 ,5 fold increase of at least one endogenous gene selected from the group consisting of IF127, ISO 15, and a combination thereof, compared to an afebrile enterovirus control level or a febrile control level.
15 Ϊ . An antibiotic for use in a . method of treatment of bacterial infection in a subjec t, characterized in that the subject has been selected to have in a blood sample at least a 1.5 fold increase of at least on endogenous gene selected from the group consisting of JTGAM and ITGAX, compared to an afebrile control level or a viral-infected control level.
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